bsai hfv2  (New England Biolabs)


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    Structured Review

    New England Biolabs bsai hfv2
    Efficiency of different <t>BsaI</t> enzymes. To test the efficiency of the <t>BsaI-HFv2</t> 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
    Bsai Hfv2, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 40 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bsai hfv2/product/New England Biolabs
    Average 97 stars, based on 40 article reviews
    Price from $9.99 to $1999.99
    bsai hfv2 - by Bioz Stars, 2022-09
    97/100 stars

    Images

    1) 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

    2) Product Images from "Development of a dedicated Golden Gate Assembly platform (RtGGA) for Rhodotorula toruloides"

    Article Title: Development of a dedicated Golden Gate Assembly platform (RtGGA) for Rhodotorula toruloides

    Journal: bioRxiv

    doi: 10.1101/2022.03.02.482697

    The RtGGA with A) Level 1 (Lv1) for the library of standardized parts−containing regions upstream (insUP) and downstream (insD) an insertional site, selection marker (M), promoters, genes, and terminators− and level 2 (Lv 2) by combining promoter (P), gene (G), terminator (T) into transcriptional units (TU). The upper part illustrates version 1.0 of Lv1 and Lv2, while the lower part illustrates version 1.1 of the RtGGA which BsaI recognition sites are flanking the parts and TUs to enable direct use of them into GGA reactions. B) Level 3 (Lv3) RtGGA multigene expression cassette and standardized overhangs from the Y. lipolytica GGA platform ( Celinska et al., 2017 ). The overhangs in italic are the changes made to make it compatible with R. toruloides C) Upper box shows the reaction method proposed in the Y. lipolytica GGA platform ( Celinska et al., 2017 ) for level 2 assembly using linearized PCR-amplified parts. The lower box shows the two-step assembly protocol adapted from Larroude et al. (2019) for the assembly of Lv2. D) Scheme of the cassettes assembled for the characterization of the promoters strength and the overexpression of the carotenoid pathway. Legend: ori – the origin of replication; black arrow– bacterial resistance gene; RFP: red fluorescence protein;
    Figure Legend Snippet: The RtGGA with A) Level 1 (Lv1) for the library of standardized parts−containing regions upstream (insUP) and downstream (insD) an insertional site, selection marker (M), promoters, genes, and terminators− and level 2 (Lv 2) by combining promoter (P), gene (G), terminator (T) into transcriptional units (TU). The upper part illustrates version 1.0 of Lv1 and Lv2, while the lower part illustrates version 1.1 of the RtGGA which BsaI recognition sites are flanking the parts and TUs to enable direct use of them into GGA reactions. B) Level 3 (Lv3) RtGGA multigene expression cassette and standardized overhangs from the Y. lipolytica GGA platform ( Celinska et al., 2017 ). The overhangs in italic are the changes made to make it compatible with R. toruloides C) Upper box shows the reaction method proposed in the Y. lipolytica GGA platform ( Celinska et al., 2017 ) for level 2 assembly using linearized PCR-amplified parts. The lower box shows the two-step assembly protocol adapted from Larroude et al. (2019) for the assembly of Lv2. D) Scheme of the cassettes assembled for the characterization of the promoters strength and the overexpression of the carotenoid pathway. Legend: ori – the origin of replication; black arrow– bacterial resistance gene; RFP: red fluorescence protein;

    Techniques Used: Selection, Marker, Expressing, Polymerase Chain Reaction, Amplification, Over Expression, Fluorescence

    3) 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

    4) 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

    5) Product Images from "Experimental and theoretical aspects of Golden Gate Assembly assays"

    Article Title: Experimental and theoretical aspects of Golden Gate Assembly assays

    Journal: bioRxiv

    doi: 10.1101/2022.09.09.507109

    Experimental flow for the Golden Gate Assembly (GGA) assay. A. First, two PCR reactions are performed on the pEX-K4-pSAM001 plasmid (4515 bp) using primers that introduce both the recognition site of the type IIS restriction endonuclease BsaI-HFv2 ( GGTGTC , asterisk) and specific sequences located downstream the recognition site (step 1). After purification of PCR products, determination of DNA concentration and visualization of DNA fragments by electrophoresis (step 2), a one-pot reaction is performed (step 3). Here, both PCR fragments ( αα ’, 565 bp and β’ β , 1678 bp) are mixed at a given molarity and stoichiometry together with BsaI-HFv2 and T4 DNA ligase. Among the fragments produced by BsaI-HFv2, only those not containing BsaI-HFv2’s recognition sites yield a stable product after ligation. Shown here are nucleotides for the TAGAAT sequence, which is a sequence of six nucleotides containing a non palindromic 4 bp overhang at position 2. Note that nucleotides at positions 1 and 6 may also play a role as they govern stacking energies. Here, and except for the GCCGCC sequence, nucleotides at positions 1 and 6 vary among possible ligation products αβ, αα ’, β’β and β’α’ . To determine the percentage of product formed Ψ β (defined as 100 × [ αβ ]/( β’β ] + [ αβ ])), we perform a quantitative and robust analysis on agarose gels (see Supplementary Figure S2). B. DNA sequences showing the local binding of primers on the plasmid ( TAGAAT sequence found in the αβ product).
    Figure Legend Snippet: Experimental flow for the Golden Gate Assembly (GGA) assay. A. First, two PCR reactions are performed on the pEX-K4-pSAM001 plasmid (4515 bp) using primers that introduce both the recognition site of the type IIS restriction endonuclease BsaI-HFv2 ( GGTGTC , asterisk) and specific sequences located downstream the recognition site (step 1). After purification of PCR products, determination of DNA concentration and visualization of DNA fragments by electrophoresis (step 2), a one-pot reaction is performed (step 3). Here, both PCR fragments ( αα ’, 565 bp and β’ β , 1678 bp) are mixed at a given molarity and stoichiometry together with BsaI-HFv2 and T4 DNA ligase. Among the fragments produced by BsaI-HFv2, only those not containing BsaI-HFv2’s recognition sites yield a stable product after ligation. Shown here are nucleotides for the TAGAAT sequence, which is a sequence of six nucleotides containing a non palindromic 4 bp overhang at position 2. Note that nucleotides at positions 1 and 6 may also play a role as they govern stacking energies. Here, and except for the GCCGCC sequence, nucleotides at positions 1 and 6 vary among possible ligation products αβ, αα ’, β’β and β’α’ . To determine the percentage of product formed Ψ β (defined as 100 × [ αβ ]/( β’β ] + [ αβ ])), we perform a quantitative and robust analysis on agarose gels (see Supplementary Figure S2). B. DNA sequences showing the local binding of primers on the plasmid ( TAGAAT sequence found in the αβ product).

    Techniques Used: Polymerase Chain Reaction, Plasmid Preparation, Introduce, Purification, Concentration Assay, Electrophoresis, Produced, Ligation, Sequencing, Binding Assay

    6) Product Images from "Experimental and theoretical aspects of Golden Gate Assembly assays"

    Article Title: Experimental and theoretical aspects of Golden Gate Assembly assays

    Journal: bioRxiv

    doi: 10.1101/2022.09.09.507109

    Experimental flow for the Golden Gate Assembly (GGA) assay. A. First, two PCR reactions are performed on the pEX-K4-pSAM001 plasmid (4515 bp) using primers that introduce both the recognition site of the type IIS restriction endonuclease BsaI-HFv2 ( GGTGTC , asterisk) and specific sequences located downstream the recognition site (step 1). After purification of PCR products, determination of DNA concentration and visualization of DNA fragments by electrophoresis (step 2), a one-pot reaction is performed (step 3). Here, both PCR fragments ( αα ’, 565 bp and β’ β , 1678 bp) are mixed at a given molarity and stoichiometry together with BsaI-HFv2 and T4 DNA ligase. Among the fragments produced by BsaI-HFv2, only those not containing BsaI-HFv2’s recognition sites yield a stable product after ligation. Shown here are nucleotides for the TAGAAT sequence, which is a sequence of six nucleotides containing a non palindromic 4 bp overhang at position 2. Note that nucleotides at positions 1 and 6 may also play a role as they govern stacking energies. Here, and except for the GCCGCC sequence, nucleotides at positions 1 and 6 vary among possible ligation products αβ, αα ’, β’β and β’α’ . To determine the percentage of product formed Ψ β (defined as 100 × [ αβ ]/( β’β ] + [ αβ ])), we perform a quantitative and robust analysis on agarose gels (see Supplementary Figure S2). B. DNA sequences showing the local binding of primers on the plasmid ( TAGAAT sequence found in the αβ product).
    Figure Legend Snippet: Experimental flow for the Golden Gate Assembly (GGA) assay. A. First, two PCR reactions are performed on the pEX-K4-pSAM001 plasmid (4515 bp) using primers that introduce both the recognition site of the type IIS restriction endonuclease BsaI-HFv2 ( GGTGTC , asterisk) and specific sequences located downstream the recognition site (step 1). After purification of PCR products, determination of DNA concentration and visualization of DNA fragments by electrophoresis (step 2), a one-pot reaction is performed (step 3). Here, both PCR fragments ( αα ’, 565 bp and β’ β , 1678 bp) are mixed at a given molarity and stoichiometry together with BsaI-HFv2 and T4 DNA ligase. Among the fragments produced by BsaI-HFv2, only those not containing BsaI-HFv2’s recognition sites yield a stable product after ligation. Shown here are nucleotides for the TAGAAT sequence, which is a sequence of six nucleotides containing a non palindromic 4 bp overhang at position 2. Note that nucleotides at positions 1 and 6 may also play a role as they govern stacking energies. Here, and except for the GCCGCC sequence, nucleotides at positions 1 and 6 vary among possible ligation products αβ, αα ’, β’β and β’α’ . To determine the percentage of product formed Ψ β (defined as 100 × [ αβ ]/( β’β ] + [ αβ ])), we perform a quantitative and robust analysis on agarose gels (see Supplementary Figure S2). B. DNA sequences showing the local binding of primers on the plasmid ( TAGAAT sequence found in the αβ product).

    Techniques Used: Polymerase Chain Reaction, Plasmid Preparation, Introduce, Purification, Concentration Assay, Electrophoresis, Produced, Ligation, Sequencing, Binding Assay

    7) Product Images from "Development of a dedicated Golden Gate Assembly platform (RtGGA) for Rhodotorula toruloides"

    Article Title: Development of a dedicated Golden Gate Assembly platform (RtGGA) for Rhodotorula toruloides

    Journal: bioRxiv

    doi: 10.1101/2022.03.02.482697

    The RtGGA with A) Level 1 (Lv1) for the library of standardized parts−containing regions upstream (insUP) and downstream (insD) an insertional site, selection marker (M), promoters, genes, and terminators− and level 2 (Lv 2) by combining promoter (P), gene (G), terminator (T) into transcriptional units (TU). The upper part illustrates version 1.0 of Lv1 and Lv2, while the lower part illustrates version 1.1 of the RtGGA which BsaI recognition sites are flanking the parts and TUs to enable direct use of them into GGA reactions. B) Level 3 (Lv3) RtGGA multigene expression cassette and standardized overhangs from the Y. lipolytica GGA platform ( Celinska et al., 2017 ). The overhangs in italic are the changes made to make it compatible with R. toruloides C) Upper box shows the reaction method proposed in the Y. lipolytica GGA platform ( Celinska et al., 2017 ) for level 2 assembly using linearized PCR-amplified parts. The lower box shows the two-step assembly protocol adapted from Larroude et al. (2019) for the assembly of Lv2. D) Scheme of the cassettes assembled for the characterization of the promoters strength and the overexpression of the carotenoid pathway. Legend: ori – the origin of replication; black arrow– bacterial resistance gene; RFP: red fluorescence protein;
    Figure Legend Snippet: The RtGGA with A) Level 1 (Lv1) for the library of standardized parts−containing regions upstream (insUP) and downstream (insD) an insertional site, selection marker (M), promoters, genes, and terminators− and level 2 (Lv 2) by combining promoter (P), gene (G), terminator (T) into transcriptional units (TU). The upper part illustrates version 1.0 of Lv1 and Lv2, while the lower part illustrates version 1.1 of the RtGGA which BsaI recognition sites are flanking the parts and TUs to enable direct use of them into GGA reactions. B) Level 3 (Lv3) RtGGA multigene expression cassette and standardized overhangs from the Y. lipolytica GGA platform ( Celinska et al., 2017 ). The overhangs in italic are the changes made to make it compatible with R. toruloides C) Upper box shows the reaction method proposed in the Y. lipolytica GGA platform ( Celinska et al., 2017 ) for level 2 assembly using linearized PCR-amplified parts. The lower box shows the two-step assembly protocol adapted from Larroude et al. (2019) for the assembly of Lv2. D) Scheme of the cassettes assembled for the characterization of the promoters strength and the overexpression of the carotenoid pathway. Legend: ori – the origin of replication; black arrow– bacterial resistance gene; RFP: red fluorescence protein;

    Techniques Used: Selection, Marker, Expressing, Polymerase Chain Reaction, Amplification, Over Expression, Fluorescence

    8) Product Images from "Enabling one-pot Golden Gate assemblies of unprecedented complexity using data-optimized assembly design"

    Article Title: Enabling one-pot Golden Gate assemblies of unprecedented complexity using data-optimized assembly design

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0238592

    Nucleotide mismatches in assembly reactions with T4 DNA ligase and Type IIS restriction enzymes generating four-base overhangs. Mismatch frequencies for assembly reactions with T4 DNA ligase and BsaI-HFv2 (blue), BsmBI-v2 (orange), Esp3I (gray), or BbsI-HF (yellow) were grouped according to nucleotide mispair (A:A, A:C, A:G, C:C, C:T, G:G, G:T, T:T). The error bars depict the range between the maximum and minimum observed mismatch frequencies for two experimental replicates.
    Figure Legend Snippet: Nucleotide mismatches in assembly reactions with T4 DNA ligase and Type IIS restriction enzymes generating four-base overhangs. Mismatch frequencies for assembly reactions with T4 DNA ligase and BsaI-HFv2 (blue), BsmBI-v2 (orange), Esp3I (gray), or BbsI-HF (yellow) were grouped according to nucleotide mispair (A:A, A:C, A:G, C:C, C:T, G:G, G:T, T:T). The error bars depict the range between the maximum and minimum observed mismatch frequencies for two experimental replicates.

    Techniques Used:

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    New England Biolabs bsai hfv2
    Efficiency of different <t>BsaI</t> enzymes. To test the efficiency of the <t>BsaI-HFv2</t> 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
    Bsai Hfv2, 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
    https://www.bioz.com/result/bsai hfv2/product/New England Biolabs
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    bsai hfv2 - by Bioz Stars, 2022-09
    97/100 stars
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    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

    Journal: BMC Biotechnology

    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

    doi: 10.1186/s12896-020-00616-z

    Figure Lengend 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

    Article Snippet: In this particular case, efficiency jumped from 0 positives out of 12 tested when the original BsaI version was used to 9 positive clones out of 12 tested for BsaI-HFv2 (Fig. ).

    Techniques: Clone Assay, Agarose Gel Electrophoresis

    The RtGGA with A) Level 1 (Lv1) for the library of standardized parts−containing regions upstream (insUP) and downstream (insD) an insertional site, selection marker (M), promoters, genes, and terminators− and level 2 (Lv 2) by combining promoter (P), gene (G), terminator (T) into transcriptional units (TU). The upper part illustrates version 1.0 of Lv1 and Lv2, while the lower part illustrates version 1.1 of the RtGGA which BsaI recognition sites are flanking the parts and TUs to enable direct use of them into GGA reactions. B) Level 3 (Lv3) RtGGA multigene expression cassette and standardized overhangs from the Y. lipolytica GGA platform ( Celinska et al., 2017 ). The overhangs in italic are the changes made to make it compatible with R. toruloides C) Upper box shows the reaction method proposed in the Y. lipolytica GGA platform ( Celinska et al., 2017 ) for level 2 assembly using linearized PCR-amplified parts. The lower box shows the two-step assembly protocol adapted from Larroude et al. (2019) for the assembly of Lv2. D) Scheme of the cassettes assembled for the characterization of the promoters strength and the overexpression of the carotenoid pathway. Legend: ori – the origin of replication; black arrow– bacterial resistance gene; RFP: red fluorescence protein;

    Journal: bioRxiv

    Article Title: Development of a dedicated Golden Gate Assembly platform (RtGGA) for Rhodotorula toruloides

    doi: 10.1101/2022.03.02.482697

    Figure Lengend Snippet: The RtGGA with A) Level 1 (Lv1) for the library of standardized parts−containing regions upstream (insUP) and downstream (insD) an insertional site, selection marker (M), promoters, genes, and terminators− and level 2 (Lv 2) by combining promoter (P), gene (G), terminator (T) into transcriptional units (TU). The upper part illustrates version 1.0 of Lv1 and Lv2, while the lower part illustrates version 1.1 of the RtGGA which BsaI recognition sites are flanking the parts and TUs to enable direct use of them into GGA reactions. B) Level 3 (Lv3) RtGGA multigene expression cassette and standardized overhangs from the Y. lipolytica GGA platform ( Celinska et al., 2017 ). The overhangs in italic are the changes made to make it compatible with R. toruloides C) Upper box shows the reaction method proposed in the Y. lipolytica GGA platform ( Celinska et al., 2017 ) for level 2 assembly using linearized PCR-amplified parts. The lower box shows the two-step assembly protocol adapted from Larroude et al. (2019) for the assembly of Lv2. D) Scheme of the cassettes assembled for the characterization of the promoters strength and the overexpression of the carotenoid pathway. Legend: ori – the origin of replication; black arrow– bacterial resistance gene; RFP: red fluorescence protein;

    Article Snippet: The reaction to assemble level 3 (Lv 3) GGA was prepared as follows: 75 ng of pGGA, the mass of each insert (TOPO™ plasmid) required for 2:1 molar ratio of insert:vector, 2 µl of T4 DNA Ligase Buffer (New England Biolabs), 1 µl of T4 or T7 DNA Ligase (New England Biolabs, Ipswich, USA), 1 µl of BsaI-HFV2 (New England Biolabs), up to 20 µl of nuclease-free H2O.

    Techniques: Selection, Marker, Expressing, Polymerase Chain Reaction, Amplification, Over Expression, Fluorescence

    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.

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gkac300

    Figure Lengend 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.

    Article Snippet: DNA construct designs were assembled by mixing PCR parts and oligonucleotide parts together with BsaI (BsaI-HFv2, NEB) and T4 DNA ligase (NEB) in 1× T4 DNA ligase buffer (NEB).

    Techniques: 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.

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gkac300

    Figure Lengend 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.

    Article Snippet: DNA construct designs were assembled by mixing PCR parts and oligonucleotide parts together with BsaI (BsaI-HFv2, NEB) and T4 DNA ligase (NEB) in 1× T4 DNA ligase buffer (NEB).

    Techniques: 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.

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gkac300

    Figure Lengend 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.

    Article Snippet: DNA construct designs were assembled by mixing PCR parts and oligonucleotide parts together with BsaI (BsaI-HFv2, NEB) and T4 DNA ligase (NEB) in 1× T4 DNA ligase buffer (NEB).

    Techniques: 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.

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gkac300

    Figure Lengend 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.

    Article Snippet: DNA construct designs were assembled by mixing PCR parts and oligonucleotide parts together with BsaI (BsaI-HFv2, NEB) and T4 DNA ligase (NEB) in 1× T4 DNA ligase buffer (NEB).

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