bsai  (New England Biolabs)


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    Structured Review

    New England Biolabs bsai
    Fabrication of torsionally-constrained DNA with length up to 10 kb by golden gate assembly. ( A ) 0.9 kbp DNA handles were made by PCR using either biotin or digoxigenin dUTP incorporation. Unlabelled PCR sections of 2.1 kb with encoded <t>BsaI</t> sites were separately made. The designed structure after joining of the purification PCR parts by golden gate assembly is 10.1 kb in length. ( B ) Agarose gel image of the product from the golden gate assembly reaction. The band at ∼10 kb reacts with both streptavidin and anti-digoxigenin indicating successful dual labelling. ( C ) Schematic of magnetic tweezers used for force spectroscopy measurements. ( D ) Example torsional response of single DNA tether showing characteristic supercoiling at low force. ( E ) Force extension curves of six DNA tethers in different colours. ( F ) Schematic of flow stretching of DNA molecules. ( G ) TIRF image of multiple beads stretched by a flow of 0.5 μl/s.
    Bsai, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 86/100, based on 40 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 86 stars, based on 40 article reviews
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    bsai - by Bioz Stars, 2022-11
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    Images

    1) Product Images from "Efficient golden gate assembly of DNA constructs for single molecule force spectroscopy and imaging"

    Article Title: Efficient golden gate assembly of DNA constructs for single molecule force spectroscopy and imaging

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkac300

    Fabrication of torsionally-constrained DNA with length up to 10 kb by golden gate assembly. ( A ) 0.9 kbp DNA handles were made by PCR using either biotin or digoxigenin dUTP incorporation. Unlabelled PCR sections of 2.1 kb with encoded BsaI sites were separately made. The designed structure after joining of the purification PCR parts by golden gate assembly is 10.1 kb in length. ( B ) Agarose gel image of the product from the golden gate assembly reaction. The band at ∼10 kb reacts with both streptavidin and anti-digoxigenin indicating successful dual labelling. ( C ) Schematic of magnetic tweezers used for force spectroscopy measurements. ( D ) Example torsional response of single DNA tether showing characteristic supercoiling at low force. ( E ) Force extension curves of six DNA tethers in different colours. ( F ) Schematic of flow stretching of DNA molecules. ( G ) TIRF image of multiple beads stretched by a flow of 0.5 μl/s.
    Figure Legend Snippet: Fabrication of torsionally-constrained DNA with length up to 10 kb by golden gate assembly. ( A ) 0.9 kbp DNA handles were made by PCR using either biotin or digoxigenin dUTP incorporation. Unlabelled PCR sections of 2.1 kb with encoded BsaI sites were separately made. The designed structure after joining of the purification PCR parts by golden gate assembly is 10.1 kb in length. ( B ) Agarose gel image of the product from the golden gate assembly reaction. The band at ∼10 kb reacts with both streptavidin and anti-digoxigenin indicating successful dual labelling. ( C ) Schematic of magnetic tweezers used for force spectroscopy measurements. ( D ) Example torsional response of single DNA tether showing characteristic supercoiling at low force. ( E ) Force extension curves of six DNA tethers in different colours. ( F ) Schematic of flow stretching of DNA molecules. ( G ) TIRF image of multiple beads stretched by a flow of 0.5 μl/s.

    Techniques Used: Polymerase Chain Reaction, Purification, Agarose Gel Electrophoresis, Spectroscopy

    Large DNA hairpin construction. ( A ) Design of DNA hairpin with 1.5 kb duplex arm and 2.1 kb spacer between the bead and surface. ( B ) Four PCR amplicons are synthesized together with two oligonucleotide parts and incubated with BsaI and T4 DNA ligase. ( C ) Gel analysis of final product. Bands from the starting material PCR amplicons, together with intermediate products and the final full-length product are indicated. ( D ) Schematic of DNA hairpin assembled in a magnetic tweezers experiment. At high forces the hairpin section is unzipped. ( E ) Example traces from three separate beads showing force–extension curves during increasing and decreasing force ramps. ( F ) Single molecule unwinding events after addition of the helicase PcrA at a constant force of 11 pN.
    Figure Legend Snippet: Large DNA hairpin construction. ( A ) Design of DNA hairpin with 1.5 kb duplex arm and 2.1 kb spacer between the bead and surface. ( B ) Four PCR amplicons are synthesized together with two oligonucleotide parts and incubated with BsaI and T4 DNA ligase. ( C ) Gel analysis of final product. Bands from the starting material PCR amplicons, together with intermediate products and the final full-length product are indicated. ( D ) Schematic of DNA hairpin assembled in a magnetic tweezers experiment. At high forces the hairpin section is unzipped. ( E ) Example traces from three separate beads showing force–extension curves during increasing and decreasing force ramps. ( F ) Single molecule unwinding events after addition of the helicase PcrA at a constant force of 11 pN.

    Techniques Used: Polymerase Chain Reaction, Synthesized, Incubation

    Construction of DNA with a short synthetic hairpin. ( A ) Design of construct with labelled biotin and digoxigenin handles and 75 bp synthetic hairpin at centre. ( B ) The design is assembled by separately preparing the PCR parts and oligo parts as described in Figure 1 before a one-pot incubation with BsaI and T4 DNA ligase. ( C ) Agarose gel image of product showing band corresponding to full-length construct. ( D ) Schematic of magnetic tweezers experiment and extension-time trace at 14 pN for one example bead. The extension shows spontaneous fluctuations between two states characteristic of the unfolding and refolding of the DNA hairpin.
    Figure Legend Snippet: Construction of DNA with a short synthetic hairpin. ( A ) Design of construct with labelled biotin and digoxigenin handles and 75 bp synthetic hairpin at centre. ( B ) The design is assembled by separately preparing the PCR parts and oligo parts as described in Figure 1 before a one-pot incubation with BsaI and T4 DNA ligase. ( C ) Agarose gel image of product showing band corresponding to full-length construct. ( D ) Schematic of magnetic tweezers experiment and extension-time trace at 14 pN for one example bead. The extension shows spontaneous fluctuations between two states characteristic of the unfolding and refolding of the DNA hairpin.

    Techniques Used: Construct, Polymerase Chain Reaction, Incubation, Agarose Gel Electrophoresis

    Workflow for generating long duplex DNA and hairpin structures via golden gate assembly. PCR amplicons are generated with primer overhangs that code for the BsaI recognition site and a unique four letter sequence generated as a 5′ overhang after BsaI digestion (each four base overhang containing sequence and its reverse complement is represented by a different colour). For attachment to surfaces or beads, labelled dUTPs are included in the amplification step. Separately, synthetic oligonucleotide parts are annealed together to form different structures such as duplexes, connectors and hairpin loops. These oligonucleotide parts have four base overhangs designed to base pair with specific PCR amplicons. To form the final construct design, specific subsets of the PCR parts and oligonucleotide parts are incubated together with BsaI and T4 DNA ligase in a one pot reaction.
    Figure Legend Snippet: Workflow for generating long duplex DNA and hairpin structures via golden gate assembly. PCR amplicons are generated with primer overhangs that code for the BsaI recognition site and a unique four letter sequence generated as a 5′ overhang after BsaI digestion (each four base overhang containing sequence and its reverse complement is represented by a different colour). For attachment to surfaces or beads, labelled dUTPs are included in the amplification step. Separately, synthetic oligonucleotide parts are annealed together to form different structures such as duplexes, connectors and hairpin loops. These oligonucleotide parts have four base overhangs designed to base pair with specific PCR amplicons. To form the final construct design, specific subsets of the PCR parts and oligonucleotide parts are incubated together with BsaI and T4 DNA ligase in a one pot reaction.

    Techniques Used: Polymerase Cycling Assembly, Generated, Sequencing, Amplification, Polymerase Chain Reaction, Construct, Incubation

    2) Product Images from "Multiple gene expression in plants using MIDAS‐P, a versatile type II restriction‐based modular expression vector). Multiple gene expression in plants using MIDAS‐P, a versatile type II restriction‐based modular expression vector"

    Article Title: Multiple gene expression in plants using MIDAS‐P, a versatile type II restriction‐based modular expression vector). Multiple gene expression in plants using MIDAS‐P, a versatile type II restriction‐based modular expression vector

    Journal: Biotechnology and Bioengineering

    doi: 10.1002/bit.28073

    Schematic representation of the MIDAS‐P assembly system for plant expression. The system consists of two entry vectors, pWHITE and pBLUE, for cloning genes of interest and alternate sub‐cloning in the binary destination (expression) vector pMIDAS. The first transcriptional unit is constructed in pWHITE and transferred into pMIDAS using the type IIS restriction enzyme BsaI. A second transcriptional unit in pBLUE can subsequently be transferred into pMIDAS using BsmBI. Further TUs can be added by alternating transfer from pWHITE and pBLUE. The inclusion of lacZα in pMIDAS and pBLUE allows blue/white screening at each stage. The destination vector pMIDAS also has right and left T‐DNA borders for Agrobacterium ‐mediated plant transformation. GOI, gene of interest; P, promoter; pA, terminator and polyA signals; SAR, scaffold attachment region; UTR, untranslated region
    Figure Legend Snippet: Schematic representation of the MIDAS‐P assembly system for plant expression. The system consists of two entry vectors, pWHITE and pBLUE, for cloning genes of interest and alternate sub‐cloning in the binary destination (expression) vector pMIDAS. The first transcriptional unit is constructed in pWHITE and transferred into pMIDAS using the type IIS restriction enzyme BsaI. A second transcriptional unit in pBLUE can subsequently be transferred into pMIDAS using BsmBI. Further TUs can be added by alternating transfer from pWHITE and pBLUE. The inclusion of lacZα in pMIDAS and pBLUE allows blue/white screening at each stage. The destination vector pMIDAS also has right and left T‐DNA borders for Agrobacterium ‐mediated plant transformation. GOI, gene of interest; P, promoter; pA, terminator and polyA signals; SAR, scaffold attachment region; UTR, untranslated region

    Techniques Used: Expressing, Clone Assay, Subcloning, Plasmid Preparation, Construct, Transformation Assay

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    New England Biolabs bsai neb sites
    Bsai Neb Sites, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs bsai
    Bsai, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs bsai hfv2
    Protocol overview for generating custom lssDNA from plasmid DNA. a. Process for generation of long, linear ssDNA. Starting from a plasmid containing the insert of interest (IOI; blue), Golden Gate assembly is used to generate an intramolecular ligation product of the IOI. Undesired reaction products like backbone (gray) and concatemer plasmids of the insert are shown (Step 1). The two strands of the plasmid are labelled as T (top) and B (bottom), referencing the preference of the enzyme BbvCI. A cleanup step using <t>BsaI-HFv2</t> and Exonuclease III removes undesired backbone reaction products (step 2). Then, circular ssDNA can be created from dsDNA using a nickase followed by exonuclease degradation (step 3). Finally, the cssDNA is linearized by Cas9 cleavage at the BbvCI recognition site, leaving a maximum of a 7 nt 5’ or 3’ scar (step 4). b. Plasmid design . The IOI (blue) is ligated into a backbone (grey) containing a high copy number ORI, a KanR resistance cassette, a BbvCI recognition site, and flanking BsaI-HFv2 recognition sites. The inset shows the sequence design of the adjacent BbvCI and BsaI-HFv2 recognition sites.
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    Protocol overview for generating custom lssDNA from plasmid DNA. a. Process for generation of long, linear ssDNA. Starting from a plasmid containing the insert of interest (IOI; blue), Golden Gate assembly is used to generate an intramolecular ligation product of the IOI. Undesired reaction products like backbone (gray) and concatemer plasmids of the insert are shown (Step 1). The two strands of the plasmid are labelled as T (top) and B (bottom), referencing the preference of the enzyme BbvCI. A cleanup step using BsaI-HFv2 and Exonuclease III removes undesired backbone reaction products (step 2). Then, circular ssDNA can be created from dsDNA using a nickase followed by exonuclease degradation (step 3). Finally, the cssDNA is linearized by Cas9 cleavage at the BbvCI recognition site, leaving a maximum of a 7 nt 5’ or 3’ scar (step 4). b. Plasmid design . The IOI (blue) is ligated into a backbone (grey) containing a high copy number ORI, a KanR resistance cassette, a BbvCI recognition site, and flanking BsaI-HFv2 recognition sites. The inset shows the sequence design of the adjacent BbvCI and BsaI-HFv2 recognition sites.

    Journal: bioRxiv

    Article Title: A method for generating user-defined circular single-stranded DNA from plasmid DNA using Golden Gate intramolecular ligation

    doi: 10.1101/2022.11.21.517425

    Figure Lengend Snippet: Protocol overview for generating custom lssDNA from plasmid DNA. a. Process for generation of long, linear ssDNA. Starting from a plasmid containing the insert of interest (IOI; blue), Golden Gate assembly is used to generate an intramolecular ligation product of the IOI. Undesired reaction products like backbone (gray) and concatemer plasmids of the insert are shown (Step 1). The two strands of the plasmid are labelled as T (top) and B (bottom), referencing the preference of the enzyme BbvCI. A cleanup step using BsaI-HFv2 and Exonuclease III removes undesired backbone reaction products (step 2). Then, circular ssDNA can be created from dsDNA using a nickase followed by exonuclease degradation (step 3). Finally, the cssDNA is linearized by Cas9 cleavage at the BbvCI recognition site, leaving a maximum of a 7 nt 5’ or 3’ scar (step 4). b. Plasmid design . The IOI (blue) is ligated into a backbone (grey) containing a high copy number ORI, a KanR resistance cassette, a BbvCI recognition site, and flanking BsaI-HFv2 recognition sites. The inset shows the sequence design of the adjacent BbvCI and BsaI-HFv2 recognition sites.

    Article Snippet: Plasmids pIS002 and pIS003 were also constructed using Gibson Assembly similarly to pIS001, except the inserts were isolated from plasmids C16Ex3 and C16Ex4, respectively, using a restriction digest with BsaI-HFv2 (NEB; catalog # R3733L) in 1X rCutSmart Buffer and subsequent gel extraction.

    Techniques: Plasmid Preparation, Ligation, Sequencing

    cssDNA productions for three separate inserts. The optimized protocol was tested on the three different plasmids that were constructed (pIS001, pIS002, and pIS003), with inserts shown in the first column. The results of each step of the process for 1 µg of pIS001 (5161 bp), pIS002 (3980 bp), and pIS003 (3867 bp) are shown. Lanes correspond to the steps in the protocol listed in Figure 1 . Lane 0: undigested plasmid DNA containing supercoiled and linearized plasmids. Lane 1a: plasmid is digested with BsaI-HFv2. Lane 1b: Golden Gate Assembly including BsaI-HFv2 and DNA ligase. Lane 2: Cleanup step following Golden Gate assembly. Lanes 3a-b: cssDNA is visible when either the Nt.BbvCI (3a) or Nb.BbvCI (3b) nickase is used for template prep. ssDNA bands do not appear as brightly as dsDNA in part because the SYBR-Safe dye used has a lower quantum yield when bound to ssDNA than dsDNA at the wavelength used for imaging.

    Journal: bioRxiv

    Article Title: A method for generating user-defined circular single-stranded DNA from plasmid DNA using Golden Gate intramolecular ligation

    doi: 10.1101/2022.11.21.517425

    Figure Lengend Snippet: cssDNA productions for three separate inserts. The optimized protocol was tested on the three different plasmids that were constructed (pIS001, pIS002, and pIS003), with inserts shown in the first column. The results of each step of the process for 1 µg of pIS001 (5161 bp), pIS002 (3980 bp), and pIS003 (3867 bp) are shown. Lanes correspond to the steps in the protocol listed in Figure 1 . Lane 0: undigested plasmid DNA containing supercoiled and linearized plasmids. Lane 1a: plasmid is digested with BsaI-HFv2. Lane 1b: Golden Gate Assembly including BsaI-HFv2 and DNA ligase. Lane 2: Cleanup step following Golden Gate assembly. Lanes 3a-b: cssDNA is visible when either the Nt.BbvCI (3a) or Nb.BbvCI (3b) nickase is used for template prep. ssDNA bands do not appear as brightly as dsDNA in part because the SYBR-Safe dye used has a lower quantum yield when bound to ssDNA than dsDNA at the wavelength used for imaging.

    Article Snippet: Plasmids pIS002 and pIS003 were also constructed using Gibson Assembly similarly to pIS001, except the inserts were isolated from plasmids C16Ex3 and C16Ex4, respectively, using a restriction digest with BsaI-HFv2 (NEB; catalog # R3733L) in 1X rCutSmart Buffer and subsequent gel extraction.

    Techniques: Construct, Plasmid Preparation, Imaging

    Optimization of the intramolecular ligation by Golden Gate Assembly and of the cleanup step. All lanes showing reaction intermediates contain a starting amount of 1 µg of pIS001 dsDNA. a . Optimization of reaction volumes of 20 µl and 100 µl and two different BsaI-HFv2 concentrations (1 µl or 3 µl of a 20 U/µl stock). Higher BsaI-HFv2 and the larger volume resulted in less linearized IOI, and lower concatemers than other conditions. b . A larger reaction volume of 200 µl resulted in similar formation of monomeric linear plasmid and formation of concatemers when 3 µl BsaI-HFv2 was included. Lanes 6 and 7 show removal of linear DNA using ExoIII. c . Identification of bands in cleanup step. The aim of this experiment was to confirm the identity of the upper bands that appeared in the cleanup step. We first performed BsaI-HFv2 digestion of pIS001 and gel extracted the IOI (red box, lane 2). Next, we performed the Golden Gate intramolecular ligation and cleanup steps on pIS001 (lanes 3-5) and the linearized IOI (lanes 6-8). Based on similar migration of the IOI specific lanes, we conclude that the upper bands were concatemers formed during Golden Gate Assembly. d . ExoIII and lambda exonucleases were tested for functionality in the cleanup step at different dilutions using Golden Gate assembly conditions optimized in panel b. Both exonucleases were able to remove linearized DNA while not appreciably degrading circularized IOI at all concentrations evaluated. e . Optimization of BsaI-HFv2 in cleanup step (1 µl or 3 µl of a 20 U/µl stock).

    Journal: bioRxiv

    Article Title: A method for generating user-defined circular single-stranded DNA from plasmid DNA using Golden Gate intramolecular ligation

    doi: 10.1101/2022.11.21.517425

    Figure Lengend Snippet: Optimization of the intramolecular ligation by Golden Gate Assembly and of the cleanup step. All lanes showing reaction intermediates contain a starting amount of 1 µg of pIS001 dsDNA. a . Optimization of reaction volumes of 20 µl and 100 µl and two different BsaI-HFv2 concentrations (1 µl or 3 µl of a 20 U/µl stock). Higher BsaI-HFv2 and the larger volume resulted in less linearized IOI, and lower concatemers than other conditions. b . A larger reaction volume of 200 µl resulted in similar formation of monomeric linear plasmid and formation of concatemers when 3 µl BsaI-HFv2 was included. Lanes 6 and 7 show removal of linear DNA using ExoIII. c . Identification of bands in cleanup step. The aim of this experiment was to confirm the identity of the upper bands that appeared in the cleanup step. We first performed BsaI-HFv2 digestion of pIS001 and gel extracted the IOI (red box, lane 2). Next, we performed the Golden Gate intramolecular ligation and cleanup steps on pIS001 (lanes 3-5) and the linearized IOI (lanes 6-8). Based on similar migration of the IOI specific lanes, we conclude that the upper bands were concatemers formed during Golden Gate Assembly. d . ExoIII and lambda exonucleases were tested for functionality in the cleanup step at different dilutions using Golden Gate assembly conditions optimized in panel b. Both exonucleases were able to remove linearized DNA while not appreciably degrading circularized IOI at all concentrations evaluated. e . Optimization of BsaI-HFv2 in cleanup step (1 µl or 3 µl of a 20 U/µl stock).

    Article Snippet: Plasmids pIS002 and pIS003 were also constructed using Gibson Assembly similarly to pIS001, except the inserts were isolated from plasmids C16Ex3 and C16Ex4, respectively, using a restriction digest with BsaI-HFv2 (NEB; catalog # R3733L) in 1X rCutSmart Buffer and subsequent gel extraction.

    Techniques: Ligation, Plasmid Preparation, Migration