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    BsaI 5 000 units
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    Category:
    Restriction Enzymes
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    New England Biolabs bsai
    BsaI
    BsaI 5 000 units
    https://www.bioz.com/result/bsai/product/New England Biolabs
    Average 99 stars, based on 20 article reviews
    Price from $9.99 to $1999.99
    bsai - by Bioz Stars, 2020-08
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    Images

    1) 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

    2) 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

    3) 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

    4) 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

    5) 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

    6) 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

    7) 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

    8) 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

    9) 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

    10) 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

    11) Product Images from "ATP-powered molecular recognition to engineer transient multivalency and self-sorting 4D hierarchical systems"

    Article Title: ATP-powered molecular recognition to engineer transient multivalency and self-sorting 4D hierarchical systems

    Journal: Nature Communications

    doi: 10.1038/s41467-020-17479-9

    ATP-fueled transient colloid assembly (TCA), and dynamic DNA surface polymerization (TSP). a Schematic illustration of DNA wrapping-induced TCA. b Time-dependent CLSM of TCA. Scale bars = 10 μm. c Schematic representation of TSP. d Time-dependent CLSM of transient DNA growth on colloidal surface. Scale bar = 5 μm; insert = 10 μm. e Time-dependent fluorescence intensity of the fluorescent shell for TCA with programmable lifetimes. Lines are guides to the eye. Error bars are standard deviations of 50 colloids. f Singlet fraction of the colloids for TCA ( d 1 b = 38 bp) and TSP. Points correspond to analysis at random places. g Thickness of the fluorescent shells for TSP and TCA at 1 h (i: TSP, ii: TCA, iii: extracted spectra of fluorescence intensities from i and ii). Scale bar, 1 μm. h Secondary DNA tile integration and surface function reconfiguration during a running TSP. The second tile with ATTO 647 was added 1 h after the Cy3 tile. CLSM before ( i ) and after ( j ) adding the ATTO 647 tile confirm the integration and reconfiguration. Scale bar = 2 μm. k Time dependent fluorescence intensities on the colloid surface. Lines are guides to the eye. Error bars are standard deviations of 50 colloids. Conditions for a : 10.0 μM dsDNA-Cy3, 12.0 μM ssDNA, 0.92 WU μL −1 T4 DNA ligase, 1.0 U μL −1 BsaI, ca. 0.167 mg mL −1 docking strand modified colloids (ca. 1.17–1.67 × 10 8 beads mL −1 ), and 50 μM ATP, 37 °C. Conditions for c : same condition as a but using dsDNA functionalized colloids bearing sticky end a’ on the docking strand to initiate a surface tethered main chain of the DySS SfNAP. All CLSM measurements were conducted consecutively with the same microscopy settings (see Supplementary Table 1 for sequences).
    Figure Legend Snippet: ATP-fueled transient colloid assembly (TCA), and dynamic DNA surface polymerization (TSP). a Schematic illustration of DNA wrapping-induced TCA. b Time-dependent CLSM of TCA. Scale bars = 10 μm. c Schematic representation of TSP. d Time-dependent CLSM of transient DNA growth on colloidal surface. Scale bar = 5 μm; insert = 10 μm. e Time-dependent fluorescence intensity of the fluorescent shell for TCA with programmable lifetimes. Lines are guides to the eye. Error bars are standard deviations of 50 colloids. f Singlet fraction of the colloids for TCA ( d 1 b = 38 bp) and TSP. Points correspond to analysis at random places. g Thickness of the fluorescent shells for TSP and TCA at 1 h (i: TSP, ii: TCA, iii: extracted spectra of fluorescence intensities from i and ii). Scale bar, 1 μm. h Secondary DNA tile integration and surface function reconfiguration during a running TSP. The second tile with ATTO 647 was added 1 h after the Cy3 tile. CLSM before ( i ) and after ( j ) adding the ATTO 647 tile confirm the integration and reconfiguration. Scale bar = 2 μm. k Time dependent fluorescence intensities on the colloid surface. Lines are guides to the eye. Error bars are standard deviations of 50 colloids. Conditions for a : 10.0 μM dsDNA-Cy3, 12.0 μM ssDNA, 0.92 WU μL −1 T4 DNA ligase, 1.0 U μL −1 BsaI, ca. 0.167 mg mL −1 docking strand modified colloids (ca. 1.17–1.67 × 10 8 beads mL −1 ), and 50 μM ATP, 37 °C. Conditions for c : same condition as a but using dsDNA functionalized colloids bearing sticky end a’ on the docking strand to initiate a surface tethered main chain of the DySS SfNAP. All CLSM measurements were conducted consecutively with the same microscopy settings (see Supplementary Table 1 for sequences).

    Techniques Used: Confocal Laser Scanning Microscopy, Fluorescence, Modification, Microscopy

    ATP-fueled transient self-sorting (TSeSo) on a multicomponent systems level. a , b Schemes representing two different self-sorting multicomponent colloidal systems. c CLSM of two ATP-fueled colloidal assemblies, TSeSo-A, operating in the same systems in parallel. Scale bar = 5 μm; insert = 10 μm. d CLSM of TSeSo-B, whereby one colloid species is driven into assembly (TCA), while the other species is encapsulated (TSP). e Time-dependent AGE of the SfNAPs formed in TSeSo-A. f Time-dependent singlet fraction of both self-sorting systems. Points correspond to analysis at random places. g Fluorescence intensity of the colloids with time for both self-sorting systems. Lines are guides to the eye. Error bars are standard deviations of 50 colloids. Conditions for a : 10.0 μM dsDNA 1 -Cy3, 12.0 μM ssDNA 1 , 10.0 μM dsDNA 2 -ATTO 647, 12.0 μM ssDNA 2 , 0.92 WU μL −1 T4 DNA ligase, 1.0 U μL −1 BsaI, ca. 0.1 mg mL −1 DoS 1 modified colloid, 0.1 mg mL −1 DoS 2 modified colloid, 83.3 μM ATP, 37 °C. Conditions for b : The same condition as a except that the DoS 1 is changed to sticky-end a’ terminated dsDNA. All CLSM measurements were conducted consecutively with the same microscopy settings (see Supplementary Table 1 for sequences).
    Figure Legend Snippet: ATP-fueled transient self-sorting (TSeSo) on a multicomponent systems level. a , b Schemes representing two different self-sorting multicomponent colloidal systems. c CLSM of two ATP-fueled colloidal assemblies, TSeSo-A, operating in the same systems in parallel. Scale bar = 5 μm; insert = 10 μm. d CLSM of TSeSo-B, whereby one colloid species is driven into assembly (TCA), while the other species is encapsulated (TSP). e Time-dependent AGE of the SfNAPs formed in TSeSo-A. f Time-dependent singlet fraction of both self-sorting systems. Points correspond to analysis at random places. g Fluorescence intensity of the colloids with time for both self-sorting systems. Lines are guides to the eye. Error bars are standard deviations of 50 colloids. Conditions for a : 10.0 μM dsDNA 1 -Cy3, 12.0 μM ssDNA 1 , 10.0 μM dsDNA 2 -ATTO 647, 12.0 μM ssDNA 2 , 0.92 WU μL −1 T4 DNA ligase, 1.0 U μL −1 BsaI, ca. 0.1 mg mL −1 DoS 1 modified colloid, 0.1 mg mL −1 DoS 2 modified colloid, 83.3 μM ATP, 37 °C. Conditions for b : The same condition as a except that the DoS 1 is changed to sticky-end a’ terminated dsDNA. All CLSM measurements were conducted consecutively with the same microscopy settings (see Supplementary Table 1 for sequences).

    Techniques Used: Confocal Laser Scanning Microscopy, Fluorescence, Modification, Microscopy

    ATP-fueled transient DySS DNA polymerization. a , b General scheme for ATP-powered transient DySS polymerization and control over lifetimes and adaptive DySS properties. The system enters into a DySS, state B, from its monomer state, state A, upon fueling with ATP, and returns to state A once the ATP is consumed. c Time-dependent AGE (2 wt. %, 90 V, 2 h) for transient DNA polymerizations with programmable lifetimes by fueling with varied ATP concentrations (0.1, 0.2, 0.6, and 1.0 mM). d Gray scale profiles from AGE at 1.0 mM ATP quantifying the transient shift of molecular weight, which is used to calculate the mass-weighted average chain length ( \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\overline {{\mathrm{bp}}}$$\end{document} bp ¯ w ) for each kinetic aliquot. e The \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\overline {{\mathrm{bp}}}$$\end{document} bp ¯ w development with time by varying the ATP concentration from 0.1 to 1.0 mM. Lines are guides to the eye. Multiple ATP injections are shown in Fig. 3 . f Lifetimes are controlled by the ATP concentration. Error bars are standard deviations of duplicate measurements. Conditions: 37 °C, 0.05 mM M1, 0.46 WU μL −1 T4 DNA ligase, 2.5 U μL −1 BsaI, and varying amounts of ATP.
    Figure Legend Snippet: ATP-fueled transient DySS DNA polymerization. a , b General scheme for ATP-powered transient DySS polymerization and control over lifetimes and adaptive DySS properties. The system enters into a DySS, state B, from its monomer state, state A, upon fueling with ATP, and returns to state A once the ATP is consumed. c Time-dependent AGE (2 wt. %, 90 V, 2 h) for transient DNA polymerizations with programmable lifetimes by fueling with varied ATP concentrations (0.1, 0.2, 0.6, and 1.0 mM). d Gray scale profiles from AGE at 1.0 mM ATP quantifying the transient shift of molecular weight, which is used to calculate the mass-weighted average chain length ( \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\overline {{\mathrm{bp}}}$$\end{document} bp ¯ w ) for each kinetic aliquot. e The \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\overline {{\mathrm{bp}}}$$\end{document} bp ¯ w development with time by varying the ATP concentration from 0.1 to 1.0 mM. Lines are guides to the eye. Multiple ATP injections are shown in Fig. 3 . f Lifetimes are controlled by the ATP concentration. Error bars are standard deviations of duplicate measurements. Conditions: 37 °C, 0.05 mM M1, 0.46 WU μL −1 T4 DNA ligase, 2.5 U μL −1 BsaI, and varying amounts of ATP.

    Techniques Used: Molecular Weight, Concentration Assay

    ATP-fueled transient sequence-defined functionalized nucleic acid oligomers and polymers (SfNAPs). a – e Combinatorial organization of transient and programmable sequence-defined oligomers composed of varying inputs with orthogonally complementary ligation sites. Phi indicates disassembly after ATP consumption. c Time-dependent AGE with d extracted gray scale profiles for the transient oligomerization of the tetramer and e the distribution profiles of all oligomers in the DySS. f – j ATP-fueled DySS transient SfNAP with transient FRET signaling function. g Logic pathways for the ligation of the inputs to achieve FRET DNA polymers. h Time-dependent AGE (2 wt.%, 90 V, 2 h) of the transient SfNAP fueled with 0.12 mM ATP. i The \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\overline {{\mathrm{bp}}}$$\end{document} bp ¯ w development with time at different ATP concentrations. Lines are guides to the eye. j Transient FRET signaling at different ATP concentrations ( λ ex = 530 nm), and repeated fuel addition for 0.05 mM ATP; insert: FRET lifetime control by changing the ATP concentration. Shaded areas and error bars correspond to standard deviations from duplicate measurements. Conditions for a : 0.05 mM dsDNA tiles in total; 0.2 mM ATP, 0.92 WU μL −1 T4 DNA ligase, 1.0 U μL −1 BsaI, 37 °C. Conditions for f – j : 8.0 μM dsDNA-A, 8.0 μM dsDNA-C, 8.0 μM ssDNA-B, 0.92 WU μL −1 T4 DNA ligase, 1.0 U μL −1 BsaI, varied concentration of ATP, 37 °C.
    Figure Legend Snippet: ATP-fueled transient sequence-defined functionalized nucleic acid oligomers and polymers (SfNAPs). a – e Combinatorial organization of transient and programmable sequence-defined oligomers composed of varying inputs with orthogonally complementary ligation sites. Phi indicates disassembly after ATP consumption. c Time-dependent AGE with d extracted gray scale profiles for the transient oligomerization of the tetramer and e the distribution profiles of all oligomers in the DySS. f – j ATP-fueled DySS transient SfNAP with transient FRET signaling function. g Logic pathways for the ligation of the inputs to achieve FRET DNA polymers. h Time-dependent AGE (2 wt.%, 90 V, 2 h) of the transient SfNAP fueled with 0.12 mM ATP. i The \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\overline {{\mathrm{bp}}}$$\end{document} bp ¯ w development with time at different ATP concentrations. Lines are guides to the eye. j Transient FRET signaling at different ATP concentrations ( λ ex = 530 nm), and repeated fuel addition for 0.05 mM ATP; insert: FRET lifetime control by changing the ATP concentration. Shaded areas and error bars correspond to standard deviations from duplicate measurements. Conditions for a : 0.05 mM dsDNA tiles in total; 0.2 mM ATP, 0.92 WU μL −1 T4 DNA ligase, 1.0 U μL −1 BsaI, 37 °C. Conditions for f – j : 8.0 μM dsDNA-A, 8.0 μM dsDNA-C, 8.0 μM ssDNA-B, 0.92 WU μL −1 T4 DNA ligase, 1.0 U μL −1 BsaI, varied concentration of ATP, 37 °C.

    Techniques Used: Sequencing, Ligation, Concentration Assay

    12) 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

    13) 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

    14) 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

    15) 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:

    16) 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

    17) 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

    18) 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

    19) 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

    20) 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

    21) 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

    22) Product Images from "A Modular Plasmid Assembly Kit for Multigene Expression, Gene Silencing and Silencing Rescue in Plants"

    Article Title: A Modular Plasmid Assembly Kit for Multigene Expression, Gene Silencing and Silencing Rescue in Plants

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0088218

    Overview of multi-level construct assembly. The basis for the toolkit is a library of Level I functional modules, consisting of promoters (Prom), amino-terminal tags (N tag), genes of interest (GOI), carboxy-terminal tags (C tag), terminators (Term) as well as miscellaneous modules (Misc). Removal of type IIS sites can be facilitated by creation of L0 fragments for mutagenesis (see Figure S3 ). Level I modules are assembled into a Level II vector backbone by BsaI cut-ligation to construct synthetic genes. Up to 5 LII synthetic genes are combined into a LIII vector backbone by BpiI cut-ligation to create a LIII binary plasmid. Higher level constructs can be created by sequentially using LII and LIII vector backbones. For instance, LIII multigene assemblies are combined together in a LII backbone to obtain LIV plasmids, while LIV multigene assemblies are again ligated into LIII backbones to create LV constructs. BsaI and BpiI cut-ligations are used in succession to assemble the inserts of one level with the backbone of the next one. Backbone vectors for each level carry a ccdB negative selection marker, as well as a different antibiotic resistance, allowing for easy selection of correctly assembled constructs. Level 0 vectors use ampicillin resistance (Amp R ), Level I vectors gentamicin (Gen R ), LII cloning-only vectors (Amp R ), LII binary vectors spectinomycin (Sp R ) and Level III binary vectors kanamycin (Kan R ).
    Figure Legend Snippet: Overview of multi-level construct assembly. The basis for the toolkit is a library of Level I functional modules, consisting of promoters (Prom), amino-terminal tags (N tag), genes of interest (GOI), carboxy-terminal tags (C tag), terminators (Term) as well as miscellaneous modules (Misc). Removal of type IIS sites can be facilitated by creation of L0 fragments for mutagenesis (see Figure S3 ). Level I modules are assembled into a Level II vector backbone by BsaI cut-ligation to construct synthetic genes. Up to 5 LII synthetic genes are combined into a LIII vector backbone by BpiI cut-ligation to create a LIII binary plasmid. Higher level constructs can be created by sequentially using LII and LIII vector backbones. For instance, LIII multigene assemblies are combined together in a LII backbone to obtain LIV plasmids, while LIV multigene assemblies are again ligated into LIII backbones to create LV constructs. BsaI and BpiI cut-ligations are used in succession to assemble the inserts of one level with the backbone of the next one. Backbone vectors for each level carry a ccdB negative selection marker, as well as a different antibiotic resistance, allowing for easy selection of correctly assembled constructs. Level 0 vectors use ampicillin resistance (Amp R ), Level I vectors gentamicin (Gen R ), LII cloning-only vectors (Amp R ), LII binary vectors spectinomycin (Sp R ) and Level III binary vectors kanamycin (Kan R ).

    Techniques Used: Construct, Functional Assay, Mutagenesis, Plasmid Preparation, Ligation, Selection, Marker, Clone Assay

    Construction of custom binary vector backbones for promoter and gene of interest analysis. Pre-assembled binary constructs are created with Esp3I-lacZ dummies. The desired LI modules are assembled together with a LI dy-Esp3I-lacZ A-B fragment instead of a specific LI A-B promoter module or a LI dy-Esp3I-lacZ C-D fragment instead of a specific LI C-D GOI module into a LII backbone by BsaI cut-ligation. The resulting LII assembly is combined with inserts from other LII constructs into the LIII final (LIII fin) backbone by BpiI cut-ligation. Into the resulting LIII binary vectors LI A-B or LI C-D modules can be directly inserted by a combined BsaI+Esp3I cut-ligation. Alternatively the LIII binary vectors can be further refined in a second step, by addition of a ccdB dummy fragment (LI dy-Esp3I-ccdB C-D) via Esp3I cut-ligation. The final version of the pre-assembled LIII binary vector backbone can be combined with custom LI A-B or LI C-D modules in one BsaI cut-ligation using ccdB based negative selection.
    Figure Legend Snippet: Construction of custom binary vector backbones for promoter and gene of interest analysis. Pre-assembled binary constructs are created with Esp3I-lacZ dummies. The desired LI modules are assembled together with a LI dy-Esp3I-lacZ A-B fragment instead of a specific LI A-B promoter module or a LI dy-Esp3I-lacZ C-D fragment instead of a specific LI C-D GOI module into a LII backbone by BsaI cut-ligation. The resulting LII assembly is combined with inserts from other LII constructs into the LIII final (LIII fin) backbone by BpiI cut-ligation. Into the resulting LIII binary vectors LI A-B or LI C-D modules can be directly inserted by a combined BsaI+Esp3I cut-ligation. Alternatively the LIII binary vectors can be further refined in a second step, by addition of a ccdB dummy fragment (LI dy-Esp3I-ccdB C-D) via Esp3I cut-ligation. The final version of the pre-assembled LIII binary vector backbone can be combined with custom LI A-B or LI C-D modules in one BsaI cut-ligation using ccdB based negative selection.

    Techniques Used: Plasmid Preparation, Construct, Ligation, Selection

    Assembly of Level II, III and IV plasmids. A) LI modules and LI dummies (LI dy) are fused with compatible BsaI overhangs into a LII backbone vector by BsaI cut-ligation. B) To create a LIII binary vector for plant expression up to five LII synthetic genes (LII 1–2, LII 2–3, LII 3–4, LII 4–5 and LII 5–6) or suitable LII dummies (LII dy) are combined with compatible BpiI overhangs into a LIII backbone by BpiI cut-ligation. C) A LIV plasmid is constructed from LIII multigene assemblies combined with LI dy fragments, which carry compatible BsaI overhangs, into a LII backbone by BsaI cut-ligation. LI fusion sites are named A to G, while LII fusion sites are numbered from 1 to 6. Triangles indicate the orientation of the restriction sites relative to the BsaI and BpiI recognition sites.
    Figure Legend Snippet: Assembly of Level II, III and IV plasmids. A) LI modules and LI dummies (LI dy) are fused with compatible BsaI overhangs into a LII backbone vector by BsaI cut-ligation. B) To create a LIII binary vector for plant expression up to five LII synthetic genes (LII 1–2, LII 2–3, LII 3–4, LII 4–5 and LII 5–6) or suitable LII dummies (LII dy) are combined with compatible BpiI overhangs into a LIII backbone by BpiI cut-ligation. C) A LIV plasmid is constructed from LIII multigene assemblies combined with LI dy fragments, which carry compatible BsaI overhangs, into a LII backbone by BsaI cut-ligation. LI fusion sites are named A to G, while LII fusion sites are numbered from 1 to 6. Triangles indicate the orientation of the restriction sites relative to the BsaI and BpiI recognition sites.

    Techniques Used: Plasmid Preparation, Ligation, Expressing, Construct

    23) 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

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    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 ( , , ).

    Expressing:

    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
    Article Snippet: .. During another especially challenging project where 15 expression cassettes were to be assembled, we compared the influence on efficiency of two BsaI enzyme variants (BsaI from New England Biolabs Cat. No. R0535S and BsaI-HFv2 also from NEB Cat. No. R3733S). .. The BsaI-HFv2 has greatly enhanced the efficiency of more challenging assemblies.

    Plasmid Preparation:

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

    Purification:

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

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    New England Biolabs bsa i
    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.
    Bsa I, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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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