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    Structured Review

    New England Biolabs bsa i hf
    GreenGate vector design and layout. A) The GreenGate cloning system uses six different types of pUC19 based entry vectors into which the individual elements are inserted and a pGreen-IIS based destination vector. Magenta scissors represent <t>Bsa</t> <t>I</t> recognition sites. In each GreenGate reaction, six modules are ligated between the left border (LB) and the right border (RB) sequences of the destination vector yielding a ready-to-use plant transformation vector with expression unit and resistance cassette. These six modules encompass a plant promoter, an N-terminal tag, a coding sequence (i.e. the gene of interest), a C-terminal tag, a plant terminator and a plant resistance cassette for selection of transgenic plants. The modules can only be ligated in the pre-defined order. B) The orderly assembly is enabled by a set of seven different overhangs. Each module is flanked at its 5′-end by the same overhang as the 3′-end of its preceding neighbor. The individual overhangs all differ from each other by at least two out of the four nucleotides. The underlined nucleotides define coding triplets to which all other coding elements have to be in frame. C) Empty entry vector. The multiple cloning site of pUC19 has been replaced by two Bsa I recognition sites (magenta scissors), the respective overhangs for each module type and a counter-selectable ccdB <t>gene.</t> <t>DNA</t> fragments can be cloned via the specific overhangs, via the Bam HI and Kpn I sites or via A-overhangs after Xcm I digestion. Plac = lac promoter, SP6 = SP6 promoter, caR = chloramphenicol acetyltransferase gene, T7 = T7 promoter, lacZ = lacZα coding sequence, ampR = beta-lactamase gene, ori = origin of replication. D) Empty destination vector. A counter-selectable ccdB -cassette has been inserted between the LB and RB sequences of pGreen-IIS, flanked by Bsa I sites, with overhangs A and G. promoter = bacterial promoter. The pSa origin of replication ( ori A. tum. ) requires the presence of the helper plasmid pSOUP in agrobacteria.
    Bsa I Hf, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "GreenGate - A Novel, Versatile, and Efficient Cloning System for Plant Transgenesis"

    Article Title: GreenGate - A Novel, Versatile, and Efficient Cloning System for Plant Transgenesis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0083043

    GreenGate vector design and layout. A) The GreenGate cloning system uses six different types of pUC19 based entry vectors into which the individual elements are inserted and a pGreen-IIS based destination vector. Magenta scissors represent Bsa I recognition sites. In each GreenGate reaction, six modules are ligated between the left border (LB) and the right border (RB) sequences of the destination vector yielding a ready-to-use plant transformation vector with expression unit and resistance cassette. These six modules encompass a plant promoter, an N-terminal tag, a coding sequence (i.e. the gene of interest), a C-terminal tag, a plant terminator and a plant resistance cassette for selection of transgenic plants. The modules can only be ligated in the pre-defined order. B) The orderly assembly is enabled by a set of seven different overhangs. Each module is flanked at its 5′-end by the same overhang as the 3′-end of its preceding neighbor. The individual overhangs all differ from each other by at least two out of the four nucleotides. The underlined nucleotides define coding triplets to which all other coding elements have to be in frame. C) Empty entry vector. The multiple cloning site of pUC19 has been replaced by two Bsa I recognition sites (magenta scissors), the respective overhangs for each module type and a counter-selectable ccdB gene. DNA fragments can be cloned via the specific overhangs, via the Bam HI and Kpn I sites or via A-overhangs after Xcm I digestion. Plac = lac promoter, SP6 = SP6 promoter, caR = chloramphenicol acetyltransferase gene, T7 = T7 promoter, lacZ = lacZα coding sequence, ampR = beta-lactamase gene, ori = origin of replication. D) Empty destination vector. A counter-selectable ccdB -cassette has been inserted between the LB and RB sequences of pGreen-IIS, flanked by Bsa I sites, with overhangs A and G. promoter = bacterial promoter. The pSa origin of replication ( ori A. tum. ) requires the presence of the helper plasmid pSOUP in agrobacteria.
    Figure Legend Snippet: GreenGate vector design and layout. A) The GreenGate cloning system uses six different types of pUC19 based entry vectors into which the individual elements are inserted and a pGreen-IIS based destination vector. Magenta scissors represent Bsa I recognition sites. In each GreenGate reaction, six modules are ligated between the left border (LB) and the right border (RB) sequences of the destination vector yielding a ready-to-use plant transformation vector with expression unit and resistance cassette. These six modules encompass a plant promoter, an N-terminal tag, a coding sequence (i.e. the gene of interest), a C-terminal tag, a plant terminator and a plant resistance cassette for selection of transgenic plants. The modules can only be ligated in the pre-defined order. B) The orderly assembly is enabled by a set of seven different overhangs. Each module is flanked at its 5′-end by the same overhang as the 3′-end of its preceding neighbor. The individual overhangs all differ from each other by at least two out of the four nucleotides. The underlined nucleotides define coding triplets to which all other coding elements have to be in frame. C) Empty entry vector. The multiple cloning site of pUC19 has been replaced by two Bsa I recognition sites (magenta scissors), the respective overhangs for each module type and a counter-selectable ccdB gene. DNA fragments can be cloned via the specific overhangs, via the Bam HI and Kpn I sites or via A-overhangs after Xcm I digestion. Plac = lac promoter, SP6 = SP6 promoter, caR = chloramphenicol acetyltransferase gene, T7 = T7 promoter, lacZ = lacZα coding sequence, ampR = beta-lactamase gene, ori = origin of replication. D) Empty destination vector. A counter-selectable ccdB -cassette has been inserted between the LB and RB sequences of pGreen-IIS, flanked by Bsa I sites, with overhangs A and G. promoter = bacterial promoter. The pSa origin of replication ( ori A. tum. ) requires the presence of the helper plasmid pSOUP in agrobacteria.

    Techniques Used: Plasmid Preparation, Clone Assay, Transformation Assay, Expressing, Sequencing, Selection, Transgenic Assay

    Multiple expression cassettes on a single T-DNA. A) The first strategy uses one additional overhang (“H” = TAGG), two adapter modules and two intermediate vectors. In a first step, two expression cassettes (“supermodules”) are assembled in parallel in two different intermediate vectors (pGGM000 and pGGN000). The Bsa I sites in the intermediate vectors are retained in the supermodule. In the second step, these two supermodules are then transferred into a destination vector via a normal GreenGate reaction. The overhang types are given in capital letters. p1/2 = promoter, n1/2 = N-terminal tag, cds1/2 = coding sequence, c1/2 = C-terminal tag, t1/2 = terminator, r1 = plant resistance, ad.1 = FH-adapter module, ad.2 = HA-adapter module. B) Fluorescence microscopy images show Nicotiana benthamiana leaves infiltrated with a construct harboring two expression cassettes on one T-DNA created via this method. The images were taken 72 hours after infiltration and 24 hours after ethanol induction (picture on the right). The first transcriptional unit drives constitutive expression of the ALCR transcription factor ( pUBQ10:B-dummy-ALCR-D-dummy:tRBCS ; pMAS:sulfR:t35S ), the second one ( pALCA:Ω-element-GFP-NLS-D-dummy:tRBCS ) of nuclear localized GFP in presence of ethanol-bound ALCR protein. C) Only one additional element is required for the second strategy. Instead of a plant resistance cassette module, the FH-adapter module from strategy #1 and an oligo duplex (orange) with unpaired H and G overhangs are used in the GreenGate reaction. The oligo duplex contains internal Bsa I sites that would result in A and G overhangs after digestion. However, digestion is blocked by methylation of the cytosine residues in the Bsa I recognition sites, since Bsa I is sensitive to methylation. After transformation of the resulting construct into bacteria, the methylation is lost during replication because no dcm site is present. Thus, after re-isolation from bacteria, the plasmid, already containing one expression cassette, can function as an empty GreenGate destination vector, releasing A and G overhangs after digestion by Bsa I and removal of the Bsa I recognition sites from the vector backbone. This process can in principle be re-iterated infinitely. The construct is finalized by using a standard plant resistance module in the last step. D) N. benthamiana leaves infiltrated with a destination vector (pTL019) carrying three transcriptional units assembled by this method. The fluorescence signal from all three individual expression cassettes, i.e. nuclear localized BFP (left), ER-localized GFP (second from left) and nuclear localized mCherry (third from left), is visible in all transformed cells. Merge shown on the right.
    Figure Legend Snippet: Multiple expression cassettes on a single T-DNA. A) The first strategy uses one additional overhang (“H” = TAGG), two adapter modules and two intermediate vectors. In a first step, two expression cassettes (“supermodules”) are assembled in parallel in two different intermediate vectors (pGGM000 and pGGN000). The Bsa I sites in the intermediate vectors are retained in the supermodule. In the second step, these two supermodules are then transferred into a destination vector via a normal GreenGate reaction. The overhang types are given in capital letters. p1/2 = promoter, n1/2 = N-terminal tag, cds1/2 = coding sequence, c1/2 = C-terminal tag, t1/2 = terminator, r1 = plant resistance, ad.1 = FH-adapter module, ad.2 = HA-adapter module. B) Fluorescence microscopy images show Nicotiana benthamiana leaves infiltrated with a construct harboring two expression cassettes on one T-DNA created via this method. The images were taken 72 hours after infiltration and 24 hours after ethanol induction (picture on the right). The first transcriptional unit drives constitutive expression of the ALCR transcription factor ( pUBQ10:B-dummy-ALCR-D-dummy:tRBCS ; pMAS:sulfR:t35S ), the second one ( pALCA:Ω-element-GFP-NLS-D-dummy:tRBCS ) of nuclear localized GFP in presence of ethanol-bound ALCR protein. C) Only one additional element is required for the second strategy. Instead of a plant resistance cassette module, the FH-adapter module from strategy #1 and an oligo duplex (orange) with unpaired H and G overhangs are used in the GreenGate reaction. The oligo duplex contains internal Bsa I sites that would result in A and G overhangs after digestion. However, digestion is blocked by methylation of the cytosine residues in the Bsa I recognition sites, since Bsa I is sensitive to methylation. After transformation of the resulting construct into bacteria, the methylation is lost during replication because no dcm site is present. Thus, after re-isolation from bacteria, the plasmid, already containing one expression cassette, can function as an empty GreenGate destination vector, releasing A and G overhangs after digestion by Bsa I and removal of the Bsa I recognition sites from the vector backbone. This process can in principle be re-iterated infinitely. The construct is finalized by using a standard plant resistance module in the last step. D) N. benthamiana leaves infiltrated with a destination vector (pTL019) carrying three transcriptional units assembled by this method. The fluorescence signal from all three individual expression cassettes, i.e. nuclear localized BFP (left), ER-localized GFP (second from left) and nuclear localized mCherry (third from left), is visible in all transformed cells. Merge shown on the right.

    Techniques Used: Expressing, Plasmid Preparation, Sequencing, Fluorescence, Microscopy, Construct, Methylation, Transformation Assay, Isolation

    The Golden Gate principle. A) Type IIS restriction endonucleases, such as Bsa I, have a distinct, non-palindromic recognition site (red) and asymmetrically cut at a precisely defined distance regardless of the local sequence (green). Bsa I for instance creates a four base 5′-overhang starting from the second nucleotide downstream of the recognition site. B) A Golden Gate style cloning system requires two types of components, a destination vector and entry vectors containing the modules to be assembled. Each vector carries two recognition sites for the type IIS endonuclease (red) flanking the counter-selective marker on the destination vector and the modules on the entry vectors, respectively. Destination and entry vectors confer different markers for bacterial selection. The sequences in purple, blue and green represent the cutting sites. C) The orientation and position of the recognition sites is such that after digestion they remain with the backbone of the entry vectors, but are excised from the destination vector along with the counter-selectable marker ( ccdB ). D) The single stranded overhangs generated by the endonuclease can anneal to complementary sequences and be covalently linked by T4 DNA ligase. During the Golden Gate reaction in the presence of endonuclease and ligase the desired final product, but also the original vectors or a plethora of side-products (one of them shown at the bottom) can be created. However, only the desired final product is resistant to further endonucleolytic cleavage, whereas all other molecules will be cut again and again and thus will disappear from the reaction over time.
    Figure Legend Snippet: The Golden Gate principle. A) Type IIS restriction endonucleases, such as Bsa I, have a distinct, non-palindromic recognition site (red) and asymmetrically cut at a precisely defined distance regardless of the local sequence (green). Bsa I for instance creates a four base 5′-overhang starting from the second nucleotide downstream of the recognition site. B) A Golden Gate style cloning system requires two types of components, a destination vector and entry vectors containing the modules to be assembled. Each vector carries two recognition sites for the type IIS endonuclease (red) flanking the counter-selective marker on the destination vector and the modules on the entry vectors, respectively. Destination and entry vectors confer different markers for bacterial selection. The sequences in purple, blue and green represent the cutting sites. C) The orientation and position of the recognition sites is such that after digestion they remain with the backbone of the entry vectors, but are excised from the destination vector along with the counter-selectable marker ( ccdB ). D) The single stranded overhangs generated by the endonuclease can anneal to complementary sequences and be covalently linked by T4 DNA ligase. During the Golden Gate reaction in the presence of endonuclease and ligase the desired final product, but also the original vectors or a plethora of side-products (one of them shown at the bottom) can be created. However, only the desired final product is resistant to further endonucleolytic cleavage, whereas all other molecules will be cut again and again and thus will disappear from the reaction over time.

    Techniques Used: Sequencing, Clone Assay, Plasmid Preparation, Marker, Selection, Generated

    2) Product Images from "GreenGate - A Novel, Versatile, and Efficient Cloning System for Plant Transgenesis"

    Article Title: GreenGate - A Novel, Versatile, and Efficient Cloning System for Plant Transgenesis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0083043

    GreenGate vector design and layout. A) The GreenGate cloning system uses six different types of pUC19 based entry vectors into which the individual elements are inserted and a pGreen-IIS based destination vector. Magenta scissors represent Bsa I recognition sites. In each GreenGate reaction, six modules are ligated between the left border (LB) and the right border (RB) sequences of the destination vector yielding a ready-to-use plant transformation vector with expression unit and resistance cassette. These six modules encompass a plant promoter, an N-terminal tag, a coding sequence (i.e. the gene of interest), a C-terminal tag, a plant terminator and a plant resistance cassette for selection of transgenic plants. The modules can only be ligated in the pre-defined order. B) The orderly assembly is enabled by a set of seven different overhangs. Each module is flanked at its 5′-end by the same overhang as the 3′-end of its preceding neighbor. The individual overhangs all differ from each other by at least two out of the four nucleotides. The underlined nucleotides define coding triplets to which all other coding elements have to be in frame. C) Empty entry vector. The multiple cloning site of pUC19 has been replaced by two Bsa I recognition sites (magenta scissors), the respective overhangs for each module type and a counter-selectable ccdB gene. DNA fragments can be cloned via the specific overhangs, via the Bam HI and Kpn I sites or via A-overhangs after Xcm I digestion. Plac = lac promoter, SP6 = SP6 promoter, caR = chloramphenicol acetyltransferase gene, T7 = T7 promoter, lacZ = lacZα coding sequence, ampR = beta-lactamase gene, ori = origin of replication. D) Empty destination vector. A counter-selectable ccdB -cassette has been inserted between the LB and RB sequences of pGreen-IIS, flanked by Bsa I sites, with overhangs A and G. promoter = bacterial promoter. The pSa origin of replication ( ori A. tum. ) requires the presence of the helper plasmid pSOUP in agrobacteria.
    Figure Legend Snippet: GreenGate vector design and layout. A) The GreenGate cloning system uses six different types of pUC19 based entry vectors into which the individual elements are inserted and a pGreen-IIS based destination vector. Magenta scissors represent Bsa I recognition sites. In each GreenGate reaction, six modules are ligated between the left border (LB) and the right border (RB) sequences of the destination vector yielding a ready-to-use plant transformation vector with expression unit and resistance cassette. These six modules encompass a plant promoter, an N-terminal tag, a coding sequence (i.e. the gene of interest), a C-terminal tag, a plant terminator and a plant resistance cassette for selection of transgenic plants. The modules can only be ligated in the pre-defined order. B) The orderly assembly is enabled by a set of seven different overhangs. Each module is flanked at its 5′-end by the same overhang as the 3′-end of its preceding neighbor. The individual overhangs all differ from each other by at least two out of the four nucleotides. The underlined nucleotides define coding triplets to which all other coding elements have to be in frame. C) Empty entry vector. The multiple cloning site of pUC19 has been replaced by two Bsa I recognition sites (magenta scissors), the respective overhangs for each module type and a counter-selectable ccdB gene. DNA fragments can be cloned via the specific overhangs, via the Bam HI and Kpn I sites or via A-overhangs after Xcm I digestion. Plac = lac promoter, SP6 = SP6 promoter, caR = chloramphenicol acetyltransferase gene, T7 = T7 promoter, lacZ = lacZα coding sequence, ampR = beta-lactamase gene, ori = origin of replication. D) Empty destination vector. A counter-selectable ccdB -cassette has been inserted between the LB and RB sequences of pGreen-IIS, flanked by Bsa I sites, with overhangs A and G. promoter = bacterial promoter. The pSa origin of replication ( ori A. tum. ) requires the presence of the helper plasmid pSOUP in agrobacteria.

    Techniques Used: Plasmid Preparation, Clone Assay, Transformation Assay, Expressing, Sequencing, Selection, Transgenic Assay

    Multiple expression cassettes on a single T-DNA. A) The first strategy uses one additional overhang (“H” = TAGG), two adapter modules and two intermediate vectors. In a first step, two expression cassettes (“supermodules”) are assembled in parallel in two different intermediate vectors (pGGM000 and pGGN000). The Bsa I sites in the intermediate vectors are retained in the supermodule. In the second step, these two supermodules are then transferred into a destination vector via a normal GreenGate reaction. The overhang types are given in capital letters. p1/2 = promoter, n1/2 = N-terminal tag, cds1/2 = coding sequence, c1/2 = C-terminal tag, t1/2 = terminator, r1 = plant resistance, ad.1 = FH-adapter module, ad.2 = HA-adapter module. B) Fluorescence microscopy images show Nicotiana benthamiana leaves infiltrated with a construct harboring two expression cassettes on one T-DNA created via this method. The images were taken 72 hours after infiltration and 24 hours after ethanol induction (picture on the right). The first transcriptional unit drives constitutive expression of the ALCR transcription factor ( pUBQ10:B-dummy-ALCR-D-dummy:tRBCS ; pMAS:sulfR:t35S ), the second one ( pALCA:Ω-element-GFP-NLS-D-dummy:tRBCS ) of nuclear localized GFP in presence of ethanol-bound ALCR protein. C) Only one additional element is required for the second strategy. Instead of a plant resistance cassette module, the FH-adapter module from strategy #1 and an oligo duplex (orange) with unpaired H and G overhangs are used in the GreenGate reaction. The oligo duplex contains internal Bsa I sites that would result in A and G overhangs after digestion. However, digestion is blocked by methylation of the cytosine residues in the Bsa I recognition sites, since Bsa I is sensitive to methylation. After transformation of the resulting construct into bacteria, the methylation is lost during replication because no dcm site is present. Thus, after re-isolation from bacteria, the plasmid, already containing one expression cassette, can function as an empty GreenGate destination vector, releasing A and G overhangs after digestion by Bsa I and removal of the Bsa I recognition sites from the vector backbone. This process can in principle be re-iterated infinitely. The construct is finalized by using a standard plant resistance module in the last step. D) N. benthamiana leaves infiltrated with a destination vector (pTL019) carrying three transcriptional units assembled by this method. The fluorescence signal from all three individual expression cassettes, i.e. nuclear localized BFP (left), ER-localized GFP (second from left) and nuclear localized mCherry (third from left), is visible in all transformed cells. Merge shown on the right.
    Figure Legend Snippet: Multiple expression cassettes on a single T-DNA. A) The first strategy uses one additional overhang (“H” = TAGG), two adapter modules and two intermediate vectors. In a first step, two expression cassettes (“supermodules”) are assembled in parallel in two different intermediate vectors (pGGM000 and pGGN000). The Bsa I sites in the intermediate vectors are retained in the supermodule. In the second step, these two supermodules are then transferred into a destination vector via a normal GreenGate reaction. The overhang types are given in capital letters. p1/2 = promoter, n1/2 = N-terminal tag, cds1/2 = coding sequence, c1/2 = C-terminal tag, t1/2 = terminator, r1 = plant resistance, ad.1 = FH-adapter module, ad.2 = HA-adapter module. B) Fluorescence microscopy images show Nicotiana benthamiana leaves infiltrated with a construct harboring two expression cassettes on one T-DNA created via this method. The images were taken 72 hours after infiltration and 24 hours after ethanol induction (picture on the right). The first transcriptional unit drives constitutive expression of the ALCR transcription factor ( pUBQ10:B-dummy-ALCR-D-dummy:tRBCS ; pMAS:sulfR:t35S ), the second one ( pALCA:Ω-element-GFP-NLS-D-dummy:tRBCS ) of nuclear localized GFP in presence of ethanol-bound ALCR protein. C) Only one additional element is required for the second strategy. Instead of a plant resistance cassette module, the FH-adapter module from strategy #1 and an oligo duplex (orange) with unpaired H and G overhangs are used in the GreenGate reaction. The oligo duplex contains internal Bsa I sites that would result in A and G overhangs after digestion. However, digestion is blocked by methylation of the cytosine residues in the Bsa I recognition sites, since Bsa I is sensitive to methylation. After transformation of the resulting construct into bacteria, the methylation is lost during replication because no dcm site is present. Thus, after re-isolation from bacteria, the plasmid, already containing one expression cassette, can function as an empty GreenGate destination vector, releasing A and G overhangs after digestion by Bsa I and removal of the Bsa I recognition sites from the vector backbone. This process can in principle be re-iterated infinitely. The construct is finalized by using a standard plant resistance module in the last step. D) N. benthamiana leaves infiltrated with a destination vector (pTL019) carrying three transcriptional units assembled by this method. The fluorescence signal from all three individual expression cassettes, i.e. nuclear localized BFP (left), ER-localized GFP (second from left) and nuclear localized mCherry (third from left), is visible in all transformed cells. Merge shown on the right.

    Techniques Used: Expressing, Plasmid Preparation, Sequencing, Fluorescence, Microscopy, Construct, Methylation, Transformation Assay, Isolation

    The Golden Gate principle. A) Type IIS restriction endonucleases, such as Bsa I, have a distinct, non-palindromic recognition site (red) and asymmetrically cut at a precisely defined distance regardless of the local sequence (green). Bsa I for instance creates a four base 5′-overhang starting from the second nucleotide downstream of the recognition site. B) A Golden Gate style cloning system requires two types of components, a destination vector and entry vectors containing the modules to be assembled. Each vector carries two recognition sites for the type IIS endonuclease (red) flanking the counter-selective marker on the destination vector and the modules on the entry vectors, respectively. Destination and entry vectors confer different markers for bacterial selection. The sequences in purple, blue and green represent the cutting sites. C) The orientation and position of the recognition sites is such that after digestion they remain with the backbone of the entry vectors, but are excised from the destination vector along with the counter-selectable marker ( ccdB ). D) The single stranded overhangs generated by the endonuclease can anneal to complementary sequences and be covalently linked by T4 DNA ligase. During the Golden Gate reaction in the presence of endonuclease and ligase the desired final product, but also the original vectors or a plethora of side-products (one of them shown at the bottom) can be created. However, only the desired final product is resistant to further endonucleolytic cleavage, whereas all other molecules will be cut again and again and thus will disappear from the reaction over time.
    Figure Legend Snippet: The Golden Gate principle. A) Type IIS restriction endonucleases, such as Bsa I, have a distinct, non-palindromic recognition site (red) and asymmetrically cut at a precisely defined distance regardless of the local sequence (green). Bsa I for instance creates a four base 5′-overhang starting from the second nucleotide downstream of the recognition site. B) A Golden Gate style cloning system requires two types of components, a destination vector and entry vectors containing the modules to be assembled. Each vector carries two recognition sites for the type IIS endonuclease (red) flanking the counter-selective marker on the destination vector and the modules on the entry vectors, respectively. Destination and entry vectors confer different markers for bacterial selection. The sequences in purple, blue and green represent the cutting sites. C) The orientation and position of the recognition sites is such that after digestion they remain with the backbone of the entry vectors, but are excised from the destination vector along with the counter-selectable marker ( ccdB ). D) The single stranded overhangs generated by the endonuclease can anneal to complementary sequences and be covalently linked by T4 DNA ligase. During the Golden Gate reaction in the presence of endonuclease and ligase the desired final product, but also the original vectors or a plethora of side-products (one of them shown at the bottom) can be created. However, only the desired final product is resistant to further endonucleolytic cleavage, whereas all other molecules will be cut again and again and thus will disappear from the reaction over time.

    Techniques Used: Sequencing, Clone Assay, Plasmid Preparation, Marker, Selection, Generated

    3) Product Images from "A high-throughput expression screening platform to optimize the production of antimicrobial peptides"

    Article Title: A high-throughput expression screening platform to optimize the production of antimicrobial peptides

    Journal: Microbial Cell Factories

    doi: 10.1186/s12934-017-0637-5

    Golden Gate assembly of the expression plasmid in a single reaction. Six donor plasmids and one acceptor plasmid were combined to form each expression plasmid by directional cloning in a restriction-ligation step using the endonuclease Bsa I and T4 DNA ligase. The overhangs produced by Bsa I are colored
    Figure Legend Snippet: Golden Gate assembly of the expression plasmid in a single reaction. Six donor plasmids and one acceptor plasmid were combined to form each expression plasmid by directional cloning in a restriction-ligation step using the endonuclease Bsa I and T4 DNA ligase. The overhangs produced by Bsa I are colored

    Techniques Used: Expressing, Plasmid Preparation, Clone Assay, Ligation, Produced

    Basic plasmid library for E. coli . The plasmid library consists of 13 donor plasmids, resulting in 27 different expression plasmids. Three promoters (T7/ lac , T5/ lac and araBAD ) were combined with a His 6 tag and either GST or Trx as a fusion partner (or no fusion partner). A thrombin cleavage site was also included in the constructs with a fusion partner. The library includes three AMP genes encoding IMPI, BR021 or the L. sericata antifungal peptide (AFP). The T7 transcriptional terminator was present in all constructs. The chosen Bsa I overhangs are shown above the elements
    Figure Legend Snippet: Basic plasmid library for E. coli . The plasmid library consists of 13 donor plasmids, resulting in 27 different expression plasmids. Three promoters (T7/ lac , T5/ lac and araBAD ) were combined with a His 6 tag and either GST or Trx as a fusion partner (or no fusion partner). A thrombin cleavage site was also included in the constructs with a fusion partner. The library includes three AMP genes encoding IMPI, BR021 or the L. sericata antifungal peptide (AFP). The T7 transcriptional terminator was present in all constructs. The chosen Bsa I overhangs are shown above the elements

    Techniques Used: Plasmid Preparation, Expressing, Construct

    Cloning efficiencies for the Golden Gate assembly of different E. coli expression plasmids. Each dot represents one cloning procedure with an individual expression plasmid. There were no significant differences in the cloning efficiencies of the different promoters ( a ), fusion partners ( b ) or proteins ( c ). d Shows the cloning efficiencies for an expression plasmid where two consecutive plasmid features were left out, here the fusion partner and the protease cleavage site. In this case, Golden Gate cloning using two short dummy fragments was compared to a workaround strategy in which one of the neighboring plasmid feature sequences (here for the His 6 tag) carries a Bsa I overhang directly complementary to the plasmid feature “protein of interest” so that no short dummy fragments are needed. As a comparison, the cloning efficiencies for the assembly of the other 18 expression plasmids, which carry all six classes of plasmid features, are also shown
    Figure Legend Snippet: Cloning efficiencies for the Golden Gate assembly of different E. coli expression plasmids. Each dot represents one cloning procedure with an individual expression plasmid. There were no significant differences in the cloning efficiencies of the different promoters ( a ), fusion partners ( b ) or proteins ( c ). d Shows the cloning efficiencies for an expression plasmid where two consecutive plasmid features were left out, here the fusion partner and the protease cleavage site. In this case, Golden Gate cloning using two short dummy fragments was compared to a workaround strategy in which one of the neighboring plasmid feature sequences (here for the His 6 tag) carries a Bsa I overhang directly complementary to the plasmid feature “protein of interest” so that no short dummy fragments are needed. As a comparison, the cloning efficiencies for the assembly of the other 18 expression plasmids, which carry all six classes of plasmid features, are also shown

    Techniques Used: Clone Assay, Expressing, Plasmid Preparation

    4) Product Images from "TrichoGate: An Improved Vector System for a Large Scale of Functional Analysis of Trichoderma Genes"

    Article Title: TrichoGate: An Improved Vector System for a Large Scale of Functional Analysis of Trichoderma Genes

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2019.02794

    Confirmation of homologous recombination event of the HygR cassette in deletion mutants by Southern blot. (A) Colony appearance growing on axenic media (PDA) under light/dark cycle. (B–D) Schematic representation of gene deletion strategy. The ORF of each targeted gene is replaced by the homologous integration of the PtrpC/ hph fragment (1.4 kb) that confers resistance to hygromycin B. (B) A 7.3 kb fragment would result for the ts1 deletion mutants when digested with Bam HI. (C) Southern blot analysis of T. virens WT strain and ts1 deletion mutants (6, 11, 12, 20, 27, 30, 31, 32, and 54) (D) A 4.5 kb fragment would result for the ts2 deletion mutants when digested with Bsa I and Hin dIII. (E) Southern blot of T. virens WT strain and ts2 deletion mutants (1, 2, 3, 4, 5, 7, 9, 22, 26, and 35). Expected fragments sizes for each deletion event along the left side of the films.
    Figure Legend Snippet: Confirmation of homologous recombination event of the HygR cassette in deletion mutants by Southern blot. (A) Colony appearance growing on axenic media (PDA) under light/dark cycle. (B–D) Schematic representation of gene deletion strategy. The ORF of each targeted gene is replaced by the homologous integration of the PtrpC/ hph fragment (1.4 kb) that confers resistance to hygromycin B. (B) A 7.3 kb fragment would result for the ts1 deletion mutants when digested with Bam HI. (C) Southern blot analysis of T. virens WT strain and ts1 deletion mutants (6, 11, 12, 20, 27, 30, 31, 32, and 54) (D) A 4.5 kb fragment would result for the ts2 deletion mutants when digested with Bsa I and Hin dIII. (E) Southern blot of T. virens WT strain and ts2 deletion mutants (1, 2, 3, 4, 5, 7, 9, 22, 26, and 35). Expected fragments sizes for each deletion event along the left side of the films.

    Techniques Used: Homologous Recombination, Southern Blot, Hi-C

    TrichoGate cloning system. Schematic representation of the TrichoGate cloning strategy to accomplish genomic modification in T. virens . (A) Implementation of TrichoGate cloning system using type II restriction enzyme ( Bsa I), target DNA sequences containing Bsa I sites in their flanking regions with unique four bases overhangs must be subcloned in linearized entry vectors (pICH41021 or pCR-Blunt) carrying the ampicillin (AmpR) or kanamycin resistance (KanR) gene for selection in E. coli , respectively. TrichoGate vectors were constructed using up to five entry vectors (pICH41021 or pCR-Blunt) and/or PCR products containing synthetic DNA molecules of interest (e.g., promoters, markers, ORFs, terminators, tags and resistance cassettes) and one of the receptor vectors (pAGM1311 or pTrichoGate-1) that carry the KanR gene for selection in E. coli . Bsa I restriction-ligation was carried out in one single microcentrifuge tube containing target DNA molecules for up to 5 h. (B) Examples of hypothetically generated vectors using Bsa I restriction/ligation reaction for functional gene analysis in T. virens (e.g., gene deletion, gene complementation, overexpression, localization and immunoprecipitation).
    Figure Legend Snippet: TrichoGate cloning system. Schematic representation of the TrichoGate cloning strategy to accomplish genomic modification in T. virens . (A) Implementation of TrichoGate cloning system using type II restriction enzyme ( Bsa I), target DNA sequences containing Bsa I sites in their flanking regions with unique four bases overhangs must be subcloned in linearized entry vectors (pICH41021 or pCR-Blunt) carrying the ampicillin (AmpR) or kanamycin resistance (KanR) gene for selection in E. coli , respectively. TrichoGate vectors were constructed using up to five entry vectors (pICH41021 or pCR-Blunt) and/or PCR products containing synthetic DNA molecules of interest (e.g., promoters, markers, ORFs, terminators, tags and resistance cassettes) and one of the receptor vectors (pAGM1311 or pTrichoGate-1) that carry the KanR gene for selection in E. coli . Bsa I restriction-ligation was carried out in one single microcentrifuge tube containing target DNA molecules for up to 5 h. (B) Examples of hypothetically generated vectors using Bsa I restriction/ligation reaction for functional gene analysis in T. virens (e.g., gene deletion, gene complementation, overexpression, localization and immunoprecipitation).

    Techniques Used: Clone Assay, Modification, Polymerase Chain Reaction, Selection, Construct, Ligation, Generated, Functional Assay, Over Expression, Immunoprecipitation

    5) Product Images from "CRISPR/Cas9 Editing of the Bacillus subtilis Genome"

    Article Title: CRISPR/Cas9 Editing of the Bacillus subtilis Genome

    Journal: Bio-protocol

    doi: 10.21769/BioProtoc.2272

    Digestion of pPB41 or pPB105 with Bsa I-HF
    Figure Legend Snippet: Digestion of pPB41 or pPB105 with Bsa I-HF

    Techniques Used:

    6) Product Images from "Type IIs restriction based combinatory modulation technique for metabolic pathway optimization"

    Article Title: Type IIs restriction based combinatory modulation technique for metabolic pathway optimization

    Journal: Microbial Cell Factories

    doi: 10.1186/s12934-017-0659-z

    Modulation and optimization of MVA pathway with TRCM technique. a Primer design for obtaining assembly parts containing degenerate RBS sequences. Primers for amplification of MVA genes were embedded with Bsa I recognition site, optimized four bp linkers, and random RBS sequence AGGAGRNNNNNN. b TRCM DNA parts for assembly. One ready-made vector part was used to assemble with five library parts. c Combinatory expression library of MVA pathway. The plasmid library contained all five genes regulated with degenerate RBSs in various combinations
    Figure Legend Snippet: Modulation and optimization of MVA pathway with TRCM technique. a Primer design for obtaining assembly parts containing degenerate RBS sequences. Primers for amplification of MVA genes were embedded with Bsa I recognition site, optimized four bp linkers, and random RBS sequence AGGAGRNNNNNN. b TRCM DNA parts for assembly. One ready-made vector part was used to assemble with five library parts. c Combinatory expression library of MVA pathway. The plasmid library contained all five genes regulated with degenerate RBSs in various combinations

    Techniques Used: Amplification, Sequencing, Plasmid Preparation, Expressing

    7) Product Images from "Efficient Generation of Recombinant Influenza A Viruses Employing a New Approach to Overcome the Genetic Instability of HA Segments"

    Article Title: Efficient Generation of Recombinant Influenza A Viruses Employing a New Approach to Overcome the Genetic Instability of HA Segments

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0116917

    Construction strategy of the low-copy pMKP ccd B reverse genetic vector to clone unstable HA segments of IAV. The resulting vector is a hybrid of pSMART-LC-Kan and the Pol-I/ ccd B/Pol-II cassette of pMP ccd B with either AarI or Bsa I or BsmB I sites for restriction/cloning of the desired insert. Briefly, the sequence representing the Pol-I/ ccd B/Pol-II cassette excised from pMP ccd B by digestion with BsrB I were blunt end phosphorylated, and ligated into the EcoR V linearized pSMART-LC-Kan plasmid to generate pMKP ccd B. The pMKP ccd B was suitable to clone and maintain the genetically unstable RT-PCR product of H9N2/SA- and H5N1/VSVRI-HA. Transcriptional terminator (T); Repressor of Primer (ROP); low copy origin of replication (Ori); kanamycin resistance gene (Kanr), ampicillin resistance gene (Ampr).
    Figure Legend Snippet: Construction strategy of the low-copy pMKP ccd B reverse genetic vector to clone unstable HA segments of IAV. The resulting vector is a hybrid of pSMART-LC-Kan and the Pol-I/ ccd B/Pol-II cassette of pMP ccd B with either AarI or Bsa I or BsmB I sites for restriction/cloning of the desired insert. Briefly, the sequence representing the Pol-I/ ccd B/Pol-II cassette excised from pMP ccd B by digestion with BsrB I were blunt end phosphorylated, and ligated into the EcoR V linearized pSMART-LC-Kan plasmid to generate pMKP ccd B. The pMKP ccd B was suitable to clone and maintain the genetically unstable RT-PCR product of H9N2/SA- and H5N1/VSVRI-HA. Transcriptional terminator (T); Repressor of Primer (ROP); low copy origin of replication (Ori); kanamycin resistance gene (Kanr), ampicillin resistance gene (Ampr).

    Techniques Used: Plasmid Preparation, Clone Assay, Sequencing, Reverse Transcription Polymerase Chain Reaction

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    New England Biolabs bsa i hf
    GreenGate vector design and layout. A) The GreenGate cloning system uses six different types of pUC19 based entry vectors into which the individual elements are inserted and a pGreen-IIS based destination vector. Magenta scissors represent <t>Bsa</t> <t>I</t> recognition sites. In each GreenGate reaction, six modules are ligated between the left border (LB) and the right border (RB) sequences of the destination vector yielding a ready-to-use plant transformation vector with expression unit and resistance cassette. These six modules encompass a plant promoter, an N-terminal tag, a coding sequence (i.e. the gene of interest), a C-terminal tag, a plant terminator and a plant resistance cassette for selection of transgenic plants. The modules can only be ligated in the pre-defined order. B) The orderly assembly is enabled by a set of seven different overhangs. Each module is flanked at its 5′-end by the same overhang as the 3′-end of its preceding neighbor. The individual overhangs all differ from each other by at least two out of the four nucleotides. The underlined nucleotides define coding triplets to which all other coding elements have to be in frame. C) Empty entry vector. The multiple cloning site of pUC19 has been replaced by two Bsa I recognition sites (magenta scissors), the respective overhangs for each module type and a counter-selectable ccdB <t>gene.</t> <t>DNA</t> fragments can be cloned via the specific overhangs, via the Bam HI and Kpn I sites or via A-overhangs after Xcm I digestion. Plac = lac promoter, SP6 = SP6 promoter, caR = chloramphenicol acetyltransferase gene, T7 = T7 promoter, lacZ = lacZα coding sequence, ampR = beta-lactamase gene, ori = origin of replication. D) Empty destination vector. A counter-selectable ccdB -cassette has been inserted between the LB and RB sequences of pGreen-IIS, flanked by Bsa I sites, with overhangs A and G. promoter = bacterial promoter. The pSa origin of replication ( ori A. tum. ) requires the presence of the helper plasmid pSOUP in agrobacteria.
    Bsa I Hf, 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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    Golden Gate assembly of the expression plasmid in a single reaction. Six donor plasmids and one acceptor plasmid were combined to form each expression plasmid by directional cloning in a restriction-ligation step using the endonuclease <t>Bsa</t> I and T4 DNA ligase. The overhangs produced by Bsa I are colored
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    GreenGate vector design and layout. A) The GreenGate cloning system uses six different types of pUC19 based entry vectors into which the individual elements are inserted and a pGreen-IIS based destination vector. Magenta scissors represent Bsa I recognition sites. In each GreenGate reaction, six modules are ligated between the left border (LB) and the right border (RB) sequences of the destination vector yielding a ready-to-use plant transformation vector with expression unit and resistance cassette. These six modules encompass a plant promoter, an N-terminal tag, a coding sequence (i.e. the gene of interest), a C-terminal tag, a plant terminator and a plant resistance cassette for selection of transgenic plants. The modules can only be ligated in the pre-defined order. B) The orderly assembly is enabled by a set of seven different overhangs. Each module is flanked at its 5′-end by the same overhang as the 3′-end of its preceding neighbor. The individual overhangs all differ from each other by at least two out of the four nucleotides. The underlined nucleotides define coding triplets to which all other coding elements have to be in frame. C) Empty entry vector. The multiple cloning site of pUC19 has been replaced by two Bsa I recognition sites (magenta scissors), the respective overhangs for each module type and a counter-selectable ccdB gene. DNA fragments can be cloned via the specific overhangs, via the Bam HI and Kpn I sites or via A-overhangs after Xcm I digestion. Plac = lac promoter, SP6 = SP6 promoter, caR = chloramphenicol acetyltransferase gene, T7 = T7 promoter, lacZ = lacZα coding sequence, ampR = beta-lactamase gene, ori = origin of replication. D) Empty destination vector. A counter-selectable ccdB -cassette has been inserted between the LB and RB sequences of pGreen-IIS, flanked by Bsa I sites, with overhangs A and G. promoter = bacterial promoter. The pSa origin of replication ( ori A. tum. ) requires the presence of the helper plasmid pSOUP in agrobacteria.

    Journal: PLoS ONE

    Article Title: GreenGate - A Novel, Versatile, and Efficient Cloning System for Plant Transgenesis

    doi: 10.1371/journal.pone.0083043

    Figure Lengend Snippet: GreenGate vector design and layout. A) The GreenGate cloning system uses six different types of pUC19 based entry vectors into which the individual elements are inserted and a pGreen-IIS based destination vector. Magenta scissors represent Bsa I recognition sites. In each GreenGate reaction, six modules are ligated between the left border (LB) and the right border (RB) sequences of the destination vector yielding a ready-to-use plant transformation vector with expression unit and resistance cassette. These six modules encompass a plant promoter, an N-terminal tag, a coding sequence (i.e. the gene of interest), a C-terminal tag, a plant terminator and a plant resistance cassette for selection of transgenic plants. The modules can only be ligated in the pre-defined order. B) The orderly assembly is enabled by a set of seven different overhangs. Each module is flanked at its 5′-end by the same overhang as the 3′-end of its preceding neighbor. The individual overhangs all differ from each other by at least two out of the four nucleotides. The underlined nucleotides define coding triplets to which all other coding elements have to be in frame. C) Empty entry vector. The multiple cloning site of pUC19 has been replaced by two Bsa I recognition sites (magenta scissors), the respective overhangs for each module type and a counter-selectable ccdB gene. DNA fragments can be cloned via the specific overhangs, via the Bam HI and Kpn I sites or via A-overhangs after Xcm I digestion. Plac = lac promoter, SP6 = SP6 promoter, caR = chloramphenicol acetyltransferase gene, T7 = T7 promoter, lacZ = lacZα coding sequence, ampR = beta-lactamase gene, ori = origin of replication. D) Empty destination vector. A counter-selectable ccdB -cassette has been inserted between the LB and RB sequences of pGreen-IIS, flanked by Bsa I sites, with overhangs A and G. promoter = bacterial promoter. The pSa origin of replication ( ori A. tum. ) requires the presence of the helper plasmid pSOUP in agrobacteria.

    Article Snippet: For the GreenGate reaction itself 1.5 µL plasmid of each of the six modules were mixed with 1 µL of the destination vector, 1.5 µL CutSmart Buffer (alternatively: FastDigest buffer), 1.5 µL ATP (10 mM), 1 µL T4 DNA ligase (30 u/µL) and 1 µL Bsa I-HF (alternatively FastDigest Eco 31I) in a total volume of 15 µL.

    Techniques: Plasmid Preparation, Clone Assay, Transformation Assay, Expressing, Sequencing, Selection, Transgenic Assay

    Multiple expression cassettes on a single T-DNA. A) The first strategy uses one additional overhang (“H” = TAGG), two adapter modules and two intermediate vectors. In a first step, two expression cassettes (“supermodules”) are assembled in parallel in two different intermediate vectors (pGGM000 and pGGN000). The Bsa I sites in the intermediate vectors are retained in the supermodule. In the second step, these two supermodules are then transferred into a destination vector via a normal GreenGate reaction. The overhang types are given in capital letters. p1/2 = promoter, n1/2 = N-terminal tag, cds1/2 = coding sequence, c1/2 = C-terminal tag, t1/2 = terminator, r1 = plant resistance, ad.1 = FH-adapter module, ad.2 = HA-adapter module. B) Fluorescence microscopy images show Nicotiana benthamiana leaves infiltrated with a construct harboring two expression cassettes on one T-DNA created via this method. The images were taken 72 hours after infiltration and 24 hours after ethanol induction (picture on the right). The first transcriptional unit drives constitutive expression of the ALCR transcription factor ( pUBQ10:B-dummy-ALCR-D-dummy:tRBCS ; pMAS:sulfR:t35S ), the second one ( pALCA:Ω-element-GFP-NLS-D-dummy:tRBCS ) of nuclear localized GFP in presence of ethanol-bound ALCR protein. C) Only one additional element is required for the second strategy. Instead of a plant resistance cassette module, the FH-adapter module from strategy #1 and an oligo duplex (orange) with unpaired H and G overhangs are used in the GreenGate reaction. The oligo duplex contains internal Bsa I sites that would result in A and G overhangs after digestion. However, digestion is blocked by methylation of the cytosine residues in the Bsa I recognition sites, since Bsa I is sensitive to methylation. After transformation of the resulting construct into bacteria, the methylation is lost during replication because no dcm site is present. Thus, after re-isolation from bacteria, the plasmid, already containing one expression cassette, can function as an empty GreenGate destination vector, releasing A and G overhangs after digestion by Bsa I and removal of the Bsa I recognition sites from the vector backbone. This process can in principle be re-iterated infinitely. The construct is finalized by using a standard plant resistance module in the last step. D) N. benthamiana leaves infiltrated with a destination vector (pTL019) carrying three transcriptional units assembled by this method. The fluorescence signal from all three individual expression cassettes, i.e. nuclear localized BFP (left), ER-localized GFP (second from left) and nuclear localized mCherry (third from left), is visible in all transformed cells. Merge shown on the right.

    Journal: PLoS ONE

    Article Title: GreenGate - A Novel, Versatile, and Efficient Cloning System for Plant Transgenesis

    doi: 10.1371/journal.pone.0083043

    Figure Lengend Snippet: Multiple expression cassettes on a single T-DNA. A) The first strategy uses one additional overhang (“H” = TAGG), two adapter modules and two intermediate vectors. In a first step, two expression cassettes (“supermodules”) are assembled in parallel in two different intermediate vectors (pGGM000 and pGGN000). The Bsa I sites in the intermediate vectors are retained in the supermodule. In the second step, these two supermodules are then transferred into a destination vector via a normal GreenGate reaction. The overhang types are given in capital letters. p1/2 = promoter, n1/2 = N-terminal tag, cds1/2 = coding sequence, c1/2 = C-terminal tag, t1/2 = terminator, r1 = plant resistance, ad.1 = FH-adapter module, ad.2 = HA-adapter module. B) Fluorescence microscopy images show Nicotiana benthamiana leaves infiltrated with a construct harboring two expression cassettes on one T-DNA created via this method. The images were taken 72 hours after infiltration and 24 hours after ethanol induction (picture on the right). The first transcriptional unit drives constitutive expression of the ALCR transcription factor ( pUBQ10:B-dummy-ALCR-D-dummy:tRBCS ; pMAS:sulfR:t35S ), the second one ( pALCA:Ω-element-GFP-NLS-D-dummy:tRBCS ) of nuclear localized GFP in presence of ethanol-bound ALCR protein. C) Only one additional element is required for the second strategy. Instead of a plant resistance cassette module, the FH-adapter module from strategy #1 and an oligo duplex (orange) with unpaired H and G overhangs are used in the GreenGate reaction. The oligo duplex contains internal Bsa I sites that would result in A and G overhangs after digestion. However, digestion is blocked by methylation of the cytosine residues in the Bsa I recognition sites, since Bsa I is sensitive to methylation. After transformation of the resulting construct into bacteria, the methylation is lost during replication because no dcm site is present. Thus, after re-isolation from bacteria, the plasmid, already containing one expression cassette, can function as an empty GreenGate destination vector, releasing A and G overhangs after digestion by Bsa I and removal of the Bsa I recognition sites from the vector backbone. This process can in principle be re-iterated infinitely. The construct is finalized by using a standard plant resistance module in the last step. D) N. benthamiana leaves infiltrated with a destination vector (pTL019) carrying three transcriptional units assembled by this method. The fluorescence signal from all three individual expression cassettes, i.e. nuclear localized BFP (left), ER-localized GFP (second from left) and nuclear localized mCherry (third from left), is visible in all transformed cells. Merge shown on the right.

    Article Snippet: For the GreenGate reaction itself 1.5 µL plasmid of each of the six modules were mixed with 1 µL of the destination vector, 1.5 µL CutSmart Buffer (alternatively: FastDigest buffer), 1.5 µL ATP (10 mM), 1 µL T4 DNA ligase (30 u/µL) and 1 µL Bsa I-HF (alternatively FastDigest Eco 31I) in a total volume of 15 µL.

    Techniques: Expressing, Plasmid Preparation, Sequencing, Fluorescence, Microscopy, Construct, Methylation, Transformation Assay, Isolation

    The Golden Gate principle. A) Type IIS restriction endonucleases, such as Bsa I, have a distinct, non-palindromic recognition site (red) and asymmetrically cut at a precisely defined distance regardless of the local sequence (green). Bsa I for instance creates a four base 5′-overhang starting from the second nucleotide downstream of the recognition site. B) A Golden Gate style cloning system requires two types of components, a destination vector and entry vectors containing the modules to be assembled. Each vector carries two recognition sites for the type IIS endonuclease (red) flanking the counter-selective marker on the destination vector and the modules on the entry vectors, respectively. Destination and entry vectors confer different markers for bacterial selection. The sequences in purple, blue and green represent the cutting sites. C) The orientation and position of the recognition sites is such that after digestion they remain with the backbone of the entry vectors, but are excised from the destination vector along with the counter-selectable marker ( ccdB ). D) The single stranded overhangs generated by the endonuclease can anneal to complementary sequences and be covalently linked by T4 DNA ligase. During the Golden Gate reaction in the presence of endonuclease and ligase the desired final product, but also the original vectors or a plethora of side-products (one of them shown at the bottom) can be created. However, only the desired final product is resistant to further endonucleolytic cleavage, whereas all other molecules will be cut again and again and thus will disappear from the reaction over time.

    Journal: PLoS ONE

    Article Title: GreenGate - A Novel, Versatile, and Efficient Cloning System for Plant Transgenesis

    doi: 10.1371/journal.pone.0083043

    Figure Lengend Snippet: The Golden Gate principle. A) Type IIS restriction endonucleases, such as Bsa I, have a distinct, non-palindromic recognition site (red) and asymmetrically cut at a precisely defined distance regardless of the local sequence (green). Bsa I for instance creates a four base 5′-overhang starting from the second nucleotide downstream of the recognition site. B) A Golden Gate style cloning system requires two types of components, a destination vector and entry vectors containing the modules to be assembled. Each vector carries two recognition sites for the type IIS endonuclease (red) flanking the counter-selective marker on the destination vector and the modules on the entry vectors, respectively. Destination and entry vectors confer different markers for bacterial selection. The sequences in purple, blue and green represent the cutting sites. C) The orientation and position of the recognition sites is such that after digestion they remain with the backbone of the entry vectors, but are excised from the destination vector along with the counter-selectable marker ( ccdB ). D) The single stranded overhangs generated by the endonuclease can anneal to complementary sequences and be covalently linked by T4 DNA ligase. During the Golden Gate reaction in the presence of endonuclease and ligase the desired final product, but also the original vectors or a plethora of side-products (one of them shown at the bottom) can be created. However, only the desired final product is resistant to further endonucleolytic cleavage, whereas all other molecules will be cut again and again and thus will disappear from the reaction over time.

    Article Snippet: For the GreenGate reaction itself 1.5 µL plasmid of each of the six modules were mixed with 1 µL of the destination vector, 1.5 µL CutSmart Buffer (alternatively: FastDigest buffer), 1.5 µL ATP (10 mM), 1 µL T4 DNA ligase (30 u/µL) and 1 µL Bsa I-HF (alternatively FastDigest Eco 31I) in a total volume of 15 µL.

    Techniques: Sequencing, Clone Assay, Plasmid Preparation, Marker, Selection, Generated

    Golden Gate assembly of the expression plasmid in a single reaction. Six donor plasmids and one acceptor plasmid were combined to form each expression plasmid by directional cloning in a restriction-ligation step using the endonuclease Bsa I and T4 DNA ligase. The overhangs produced by Bsa I are colored

    Journal: Microbial Cell Factories

    Article Title: A high-throughput expression screening platform to optimize the production of antimicrobial peptides

    doi: 10.1186/s12934-017-0637-5

    Figure Lengend Snippet: Golden Gate assembly of the expression plasmid in a single reaction. Six donor plasmids and one acceptor plasmid were combined to form each expression plasmid by directional cloning in a restriction-ligation step using the endonuclease Bsa I and T4 DNA ligase. The overhangs produced by Bsa I are colored

    Article Snippet: The better performance of the wild-type enzyme may be caused by its full activity at 50 °C during the final restriction step, in contrast to the depleted activity of Bsa I-HF (NEB, personal communication).

    Techniques: Expressing, Plasmid Preparation, Clone Assay, Ligation, Produced

    Basic plasmid library for E. coli . The plasmid library consists of 13 donor plasmids, resulting in 27 different expression plasmids. Three promoters (T7/ lac , T5/ lac and araBAD ) were combined with a His 6 tag and either GST or Trx as a fusion partner (or no fusion partner). A thrombin cleavage site was also included in the constructs with a fusion partner. The library includes three AMP genes encoding IMPI, BR021 or the L. sericata antifungal peptide (AFP). The T7 transcriptional terminator was present in all constructs. The chosen Bsa I overhangs are shown above the elements

    Journal: Microbial Cell Factories

    Article Title: A high-throughput expression screening platform to optimize the production of antimicrobial peptides

    doi: 10.1186/s12934-017-0637-5

    Figure Lengend Snippet: Basic plasmid library for E. coli . The plasmid library consists of 13 donor plasmids, resulting in 27 different expression plasmids. Three promoters (T7/ lac , T5/ lac and araBAD ) were combined with a His 6 tag and either GST or Trx as a fusion partner (or no fusion partner). A thrombin cleavage site was also included in the constructs with a fusion partner. The library includes three AMP genes encoding IMPI, BR021 or the L. sericata antifungal peptide (AFP). The T7 transcriptional terminator was present in all constructs. The chosen Bsa I overhangs are shown above the elements

    Article Snippet: The better performance of the wild-type enzyme may be caused by its full activity at 50 °C during the final restriction step, in contrast to the depleted activity of Bsa I-HF (NEB, personal communication).

    Techniques: Plasmid Preparation, Expressing, Construct

    Cloning efficiencies for the Golden Gate assembly of different E. coli expression plasmids. Each dot represents one cloning procedure with an individual expression plasmid. There were no significant differences in the cloning efficiencies of the different promoters ( a ), fusion partners ( b ) or proteins ( c ). d Shows the cloning efficiencies for an expression plasmid where two consecutive plasmid features were left out, here the fusion partner and the protease cleavage site. In this case, Golden Gate cloning using two short dummy fragments was compared to a workaround strategy in which one of the neighboring plasmid feature sequences (here for the His 6 tag) carries a Bsa I overhang directly complementary to the plasmid feature “protein of interest” so that no short dummy fragments are needed. As a comparison, the cloning efficiencies for the assembly of the other 18 expression plasmids, which carry all six classes of plasmid features, are also shown

    Journal: Microbial Cell Factories

    Article Title: A high-throughput expression screening platform to optimize the production of antimicrobial peptides

    doi: 10.1186/s12934-017-0637-5

    Figure Lengend Snippet: Cloning efficiencies for the Golden Gate assembly of different E. coli expression plasmids. Each dot represents one cloning procedure with an individual expression plasmid. There were no significant differences in the cloning efficiencies of the different promoters ( a ), fusion partners ( b ) or proteins ( c ). d Shows the cloning efficiencies for an expression plasmid where two consecutive plasmid features were left out, here the fusion partner and the protease cleavage site. In this case, Golden Gate cloning using two short dummy fragments was compared to a workaround strategy in which one of the neighboring plasmid feature sequences (here for the His 6 tag) carries a Bsa I overhang directly complementary to the plasmid feature “protein of interest” so that no short dummy fragments are needed. As a comparison, the cloning efficiencies for the assembly of the other 18 expression plasmids, which carry all six classes of plasmid features, are also shown

    Article Snippet: The better performance of the wild-type enzyme may be caused by its full activity at 50 °C during the final restriction step, in contrast to the depleted activity of Bsa I-HF (NEB, personal communication).

    Techniques: Clone Assay, Expressing, Plasmid Preparation

    Golden Gate assembly of the expression plasmid in a single reaction. Six donor plasmids and one acceptor plasmid were combined to form each expression plasmid by directional cloning in a restriction-ligation step using the endonuclease Bsa I and T4 DNA ligase. The overhangs produced by Bsa I are colored

    Journal: Microbial Cell Factories

    Article Title: A high-throughput expression screening platform to optimize the production of antimicrobial peptides

    doi: 10.1186/s12934-017-0637-5

    Figure Lengend Snippet: Golden Gate assembly of the expression plasmid in a single reaction. Six donor plasmids and one acceptor plasmid were combined to form each expression plasmid by directional cloning in a restriction-ligation step using the endonuclease Bsa I and T4 DNA ligase. The overhangs produced by Bsa I are colored

    Article Snippet: Golden Gate cloning Each Golden Gate cloning reaction required 100 ng of the acceptor plasmid (pJ915-lacZ), 100 ng of each donor plasmid, 2.5 U Bsa I/Bsa I-HF and 300 U T4 DNA ligase (NEB, 2000 U/µL) in a reaction mixture of 15 µL in 1× T4 DNA ligation buffer (NEB).

    Techniques: Expressing, Plasmid Preparation, Clone Assay, Ligation, Produced

    Basic plasmid library for E. coli . The plasmid library consists of 13 donor plasmids, resulting in 27 different expression plasmids. Three promoters (T7/ lac , T5/ lac and araBAD ) were combined with a His 6 tag and either GST or Trx as a fusion partner (or no fusion partner). A thrombin cleavage site was also included in the constructs with a fusion partner. The library includes three AMP genes encoding IMPI, BR021 or the L. sericata antifungal peptide (AFP). The T7 transcriptional terminator was present in all constructs. The chosen Bsa I overhangs are shown above the elements

    Journal: Microbial Cell Factories

    Article Title: A high-throughput expression screening platform to optimize the production of antimicrobial peptides

    doi: 10.1186/s12934-017-0637-5

    Figure Lengend Snippet: Basic plasmid library for E. coli . The plasmid library consists of 13 donor plasmids, resulting in 27 different expression plasmids. Three promoters (T7/ lac , T5/ lac and araBAD ) were combined with a His 6 tag and either GST or Trx as a fusion partner (or no fusion partner). A thrombin cleavage site was also included in the constructs with a fusion partner. The library includes three AMP genes encoding IMPI, BR021 or the L. sericata antifungal peptide (AFP). The T7 transcriptional terminator was present in all constructs. The chosen Bsa I overhangs are shown above the elements

    Article Snippet: Golden Gate cloning Each Golden Gate cloning reaction required 100 ng of the acceptor plasmid (pJ915-lacZ), 100 ng of each donor plasmid, 2.5 U Bsa I/Bsa I-HF and 300 U T4 DNA ligase (NEB, 2000 U/µL) in a reaction mixture of 15 µL in 1× T4 DNA ligation buffer (NEB).

    Techniques: Plasmid Preparation, Expressing, Construct

    Cloning efficiencies for the Golden Gate assembly of different E. coli expression plasmids. Each dot represents one cloning procedure with an individual expression plasmid. There were no significant differences in the cloning efficiencies of the different promoters ( a ), fusion partners ( b ) or proteins ( c ). d Shows the cloning efficiencies for an expression plasmid where two consecutive plasmid features were left out, here the fusion partner and the protease cleavage site. In this case, Golden Gate cloning using two short dummy fragments was compared to a workaround strategy in which one of the neighboring plasmid feature sequences (here for the His 6 tag) carries a Bsa I overhang directly complementary to the plasmid feature “protein of interest” so that no short dummy fragments are needed. As a comparison, the cloning efficiencies for the assembly of the other 18 expression plasmids, which carry all six classes of plasmid features, are also shown

    Journal: Microbial Cell Factories

    Article Title: A high-throughput expression screening platform to optimize the production of antimicrobial peptides

    doi: 10.1186/s12934-017-0637-5

    Figure Lengend Snippet: Cloning efficiencies for the Golden Gate assembly of different E. coli expression plasmids. Each dot represents one cloning procedure with an individual expression plasmid. There were no significant differences in the cloning efficiencies of the different promoters ( a ), fusion partners ( b ) or proteins ( c ). d Shows the cloning efficiencies for an expression plasmid where two consecutive plasmid features were left out, here the fusion partner and the protease cleavage site. In this case, Golden Gate cloning using two short dummy fragments was compared to a workaround strategy in which one of the neighboring plasmid feature sequences (here for the His 6 tag) carries a Bsa I overhang directly complementary to the plasmid feature “protein of interest” so that no short dummy fragments are needed. As a comparison, the cloning efficiencies for the assembly of the other 18 expression plasmids, which carry all six classes of plasmid features, are also shown

    Article Snippet: Golden Gate cloning Each Golden Gate cloning reaction required 100 ng of the acceptor plasmid (pJ915-lacZ), 100 ng of each donor plasmid, 2.5 U Bsa I/Bsa I-HF and 300 U T4 DNA ligase (NEB, 2000 U/µL) in a reaction mixture of 15 µL in 1× T4 DNA ligation buffer (NEB).

    Techniques: Clone Assay, Expressing, Plasmid Preparation