e coli neb 5 alpha  (New England Biolabs)


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    NEB 5 alpha Competent E coli High Efficiency
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    NEB 5 alpha Competent E coli High Efficiency 20x0 05 ml
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    New England Biolabs e coli neb 5 alpha
    NEB 5 alpha Competent E coli High Efficiency
    NEB 5 alpha Competent E coli High Efficiency 20x0 05 ml
    https://www.bioz.com/result/e coli neb 5 alpha/product/New England Biolabs
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    Images

    1) Product Images from "In Vivo Selection of a Missense Mutation in adeR and Conversion of the Novel blaOXA-164 Gene into blaOXA-58 in Carbapenem-Resistant Acinetobacter baumannii Isolates from a Hospitalized Patient ▿"

    Article Title: In Vivo Selection of a Missense Mutation in adeR and Conversion of the Novel blaOXA-164 Gene into blaOXA-58 in Carbapenem-Resistant Acinetobacter baumannii Isolates from a Hospitalized Patient ▿

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.00598-10

    Imipenem and meropenem Etests for E. coli NEB 5-alpha carrying pWH1266:: bla OXA-164 and showing imipenem and meropenem heteroresistance.
    Figure Legend Snippet: Imipenem and meropenem Etests for E. coli NEB 5-alpha carrying pWH1266:: bla OXA-164 and showing imipenem and meropenem heteroresistance.

    Techniques Used:

    2) Product Images from "ZeBRα a universal, multi-fragment DNA-assembly-system with minimal hands-on time requirement"

    Article Title: ZeBRα a universal, multi-fragment DNA-assembly-system with minimal hands-on time requirement

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-39768-0

    Three or four fragments can efficiently be assembled with PPY-cell extracts, while iVEC/“transformation-cloning” with three fragments is markedly less efficient. ( a ) The assembly of four fragments in a single reaction reduces the number of successful assemblies by a factor of two to ten as compared to the three-fragment-assembly. Cell-extracts were prepared from autoinduced PPY cells and the assemblies were purified prior transformation. ( b ) Assembly of three fragments by iVEC/“transformation-cloning” with NEB 5-alpha resulted in roughly 250 recombinant colonies/µg transformed DNA. A significant number of colonies harboring plasmids with defective inserts (grey bar) and PCR-template carry-over (dotted bar) were present on the plates.
    Figure Legend Snippet: Three or four fragments can efficiently be assembled with PPY-cell extracts, while iVEC/“transformation-cloning” with three fragments is markedly less efficient. ( a ) The assembly of four fragments in a single reaction reduces the number of successful assemblies by a factor of two to ten as compared to the three-fragment-assembly. Cell-extracts were prepared from autoinduced PPY cells and the assemblies were purified prior transformation. ( b ) Assembly of three fragments by iVEC/“transformation-cloning” with NEB 5-alpha resulted in roughly 250 recombinant colonies/µg transformed DNA. A significant number of colonies harboring plasmids with defective inserts (grey bar) and PCR-template carry-over (dotted bar) were present on the plates.

    Techniques Used: Clone Assay, Purification, Transformation Assay, Recombinant, Polymerase Chain Reaction

    Strategy for SLiCE optimization and evaluation. ( a ) Flow chart of the optimization process for generating a recombinogenic E. coli lysate. The PPY strain is a DH10B-derivative used to prepare the recombinogenic cell lysate and expresses the coding sequences for Redαβγ. The extracts, derived from arabinose autoinduced PPY-cells, were compared to extracts made from non-induced PPY-cells. ( b ) Structure of the examined non-ionic detergents used to prepare the recombinogenic PPY-extracts, CHAPS, Sulfo-Betain (SB-12), n-Octyl-β-D-thioglucopyranosid (OTG), n-Octyl-β-D-glucopyranosid (OG) Dodecyl-β-D-maltosid (DDM). ( c ) PPY-extracts were tested for their recombination capacity by assembling three DNA fragments with overlapping ends, to generate a recombinant plasmid constitutively expressing a blue chromoprotein. To examine the effects detergents had on the transformation, samples were split after the assembly reaction. One part was transformed into NEB 5-alpha unpurified; the other fraction was purified by silica-column chromatography prior to transformation.
    Figure Legend Snippet: Strategy for SLiCE optimization and evaluation. ( a ) Flow chart of the optimization process for generating a recombinogenic E. coli lysate. The PPY strain is a DH10B-derivative used to prepare the recombinogenic cell lysate and expresses the coding sequences for Redαβγ. The extracts, derived from arabinose autoinduced PPY-cells, were compared to extracts made from non-induced PPY-cells. ( b ) Structure of the examined non-ionic detergents used to prepare the recombinogenic PPY-extracts, CHAPS, Sulfo-Betain (SB-12), n-Octyl-β-D-thioglucopyranosid (OTG), n-Octyl-β-D-glucopyranosid (OG) Dodecyl-β-D-maltosid (DDM). ( c ) PPY-extracts were tested for their recombination capacity by assembling three DNA fragments with overlapping ends, to generate a recombinant plasmid constitutively expressing a blue chromoprotein. To examine the effects detergents had on the transformation, samples were split after the assembly reaction. One part was transformed into NEB 5-alpha unpurified; the other fraction was purified by silica-column chromatography prior to transformation.

    Techniques Used: Flow Cytometry, Derivative Assay, Recombinant, Plasmid Preparation, Expressing, Transformation Assay, Purification, Column Chromatography

    Comparison of the influence of detergent, autoinduction, post-assembly purification and competency of used bacteria on DNA assembling efficiency. ( a ) In four of the five PPY lysis conditions induction of Redα had moderate effects. Five different detergents were tested on PPY-cells grown with either lactose (Redα un-induced) or arabinose (Redα induced). All assemblies were column-purified before transformation into NEB 5-alpha. Bars indicate standard error of three independent replicates of an assembly reaction in all following graphs. PPY recombinogenic capacity was assessed in three-fragment assemblies. ( b ) Column-purification of the three-fragment-assembly reactions led to markedly increased number of recombinant colonies for all tested detergents. In the case of CHAPS and SB-12, unpurified samples resulted in no colonies. The OTG derived PPY-extract resulted in the highest number of recombinant colonies without purification. All assemblies were arabinose-induced. ( c ) Chemical competency has profound influence on recombination efficiency. OTG prepared PPY-lysate was used in a three-fragment-assembly and transformed into commercial NEB 5-alpha competent E. coli (1 × 10 9 cfu/µg pUC DNA) or the same strain prepared by the Inoue-method 21 (2.3 × 10 6 cfu/µg DNA). ( d ) For convenient readout of the potency of the PPY-extracts PCR-fragments used for the three- and four-way assembly reactions consisted of a blue chromoprotein coding ORF, a kanamycin resistance gene an origin-of-replication (on one fragment for the three-fragment assembly) and a bacterial basal-promoter-fragment. Only successful recombinants could produce blue colonies on kanamycin plates. The PCR-fragments to be assembled had overlapping bases that summed up to about 15 bp overlapping ends.
    Figure Legend Snippet: Comparison of the influence of detergent, autoinduction, post-assembly purification and competency of used bacteria on DNA assembling efficiency. ( a ) In four of the five PPY lysis conditions induction of Redα had moderate effects. Five different detergents were tested on PPY-cells grown with either lactose (Redα un-induced) or arabinose (Redα induced). All assemblies were column-purified before transformation into NEB 5-alpha. Bars indicate standard error of three independent replicates of an assembly reaction in all following graphs. PPY recombinogenic capacity was assessed in three-fragment assemblies. ( b ) Column-purification of the three-fragment-assembly reactions led to markedly increased number of recombinant colonies for all tested detergents. In the case of CHAPS and SB-12, unpurified samples resulted in no colonies. The OTG derived PPY-extract resulted in the highest number of recombinant colonies without purification. All assemblies were arabinose-induced. ( c ) Chemical competency has profound influence on recombination efficiency. OTG prepared PPY-lysate was used in a three-fragment-assembly and transformed into commercial NEB 5-alpha competent E. coli (1 × 10 9 cfu/µg pUC DNA) or the same strain prepared by the Inoue-method 21 (2.3 × 10 6 cfu/µg DNA). ( d ) For convenient readout of the potency of the PPY-extracts PCR-fragments used for the three- and four-way assembly reactions consisted of a blue chromoprotein coding ORF, a kanamycin resistance gene an origin-of-replication (on one fragment for the three-fragment assembly) and a bacterial basal-promoter-fragment. Only successful recombinants could produce blue colonies on kanamycin plates. The PCR-fragments to be assembled had overlapping bases that summed up to about 15 bp overlapping ends.

    Techniques Used: Purification, Lysis, Transformation Assay, Recombinant, Derivative Assay, Polymerase Chain Reaction

    The recombinogenic capacity of OTG extracts from autoinduced PPY and NEB 5-alpha extracts are equivalent or better than PPY-extracts generated by the original protocol. ( a ) Plasmid map of pT7- Hin dIII- ccdB used to assess three-fragment ZeBRα assemblies. Two Hin dIII and two Bsa I sites flank the toxic-placeholder- ccdB , allowing linearization and removal of ccdB . Unique sites are available on either side of ccdB . Chloramphenicol-acetyl-transferase coding gene ( CmR ), is part of the placeholder cassette and prevents ccdB -loss during plasmid propagation. The hatched region encompasses the fragment removed during cloning. ( b ) Map of the vector pT7-GFP antisense resulting from the three-fragment test-assembly of the pT7- Hin dIII- ccdB as recipient for a GFP-ORF and a bacterial promoter containing PCR-fragment, to evaluate the efficacy of the ZeBRα-procedure. Criss-cross lines mark the fusion-sites of the assembled fragments. ( c ) Comparison of the recombination capacity of extracts prepared with OTG or CelLyticB TM from manually induced and autoinduced (denoted as “auto” in the column) PPY-cells and NEB 5-alpha. The iVEC/“transformation-cloning” of the respective fragments is shown as last column. ( d ) Green fluorescent NEB 5-alpha colonies harboring the constitutively GFP-expressing vector pT7-GFP antisense.
    Figure Legend Snippet: The recombinogenic capacity of OTG extracts from autoinduced PPY and NEB 5-alpha extracts are equivalent or better than PPY-extracts generated by the original protocol. ( a ) Plasmid map of pT7- Hin dIII- ccdB used to assess three-fragment ZeBRα assemblies. Two Hin dIII and two Bsa I sites flank the toxic-placeholder- ccdB , allowing linearization and removal of ccdB . Unique sites are available on either side of ccdB . Chloramphenicol-acetyl-transferase coding gene ( CmR ), is part of the placeholder cassette and prevents ccdB -loss during plasmid propagation. The hatched region encompasses the fragment removed during cloning. ( b ) Map of the vector pT7-GFP antisense resulting from the three-fragment test-assembly of the pT7- Hin dIII- ccdB as recipient for a GFP-ORF and a bacterial promoter containing PCR-fragment, to evaluate the efficacy of the ZeBRα-procedure. Criss-cross lines mark the fusion-sites of the assembled fragments. ( c ) Comparison of the recombination capacity of extracts prepared with OTG or CelLyticB TM from manually induced and autoinduced (denoted as “auto” in the column) PPY-cells and NEB 5-alpha. The iVEC/“transformation-cloning” of the respective fragments is shown as last column. ( d ) Green fluorescent NEB 5-alpha colonies harboring the constitutively GFP-expressing vector pT7-GFP antisense.

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

    Rendering the CcdB in the placeholder non-toxic increases the number of GFP – background colonies markedly, showing that ccdB is an essential element if working with non-gel purified vector. ( a ) The pT7- Hin dIII-dead- ccdB differs by a four base-pair deletion in the ccdB -coding sequence from it’s predecessor pT7- Hin dIII- ccdB . The dotted lines encompass the region removed during cloning. ( b ) Sequence alignment of the region encompassing the small deletion in the ccdB -ORF, in pT7- Hin dIII-dead- ccdB compared to the region in pT7- Hin dIII- ccdB and the resulting frameshift rendering the ΔCcdB non-toxic for E. coli NEB 5-alpha. The numbers denote bases in the vector. ( c ) The three-fragment assembly as shown in Fig. 6c uses the pT7-HindIII-dead- ccdB vector and OTG-derived PPY-extract to assemble pT7-GFP antisense analogous to the assemblies shown in Fig. 5 . The percentage of GFP + colonies drops from nearly 100% for pT7- Hin dIII- ccdB to 57% for pT7- Hin dIII-dead- ccdB . ( d ) Image of the mixture of GFP + and GFP − resulting from the assembly (100 µl outgrowth medium spread). The red arrow points at a cluster of GFP − colonies representing un-digested vector that would have to be screened for in a non-model assembly.
    Figure Legend Snippet: Rendering the CcdB in the placeholder non-toxic increases the number of GFP – background colonies markedly, showing that ccdB is an essential element if working with non-gel purified vector. ( a ) The pT7- Hin dIII-dead- ccdB differs by a four base-pair deletion in the ccdB -coding sequence from it’s predecessor pT7- Hin dIII- ccdB . The dotted lines encompass the region removed during cloning. ( b ) Sequence alignment of the region encompassing the small deletion in the ccdB -ORF, in pT7- Hin dIII-dead- ccdB compared to the region in pT7- Hin dIII- ccdB and the resulting frameshift rendering the ΔCcdB non-toxic for E. coli NEB 5-alpha. The numbers denote bases in the vector. ( c ) The three-fragment assembly as shown in Fig. 6c uses the pT7-HindIII-dead- ccdB vector and OTG-derived PPY-extract to assemble pT7-GFP antisense analogous to the assemblies shown in Fig. 5 . The percentage of GFP + colonies drops from nearly 100% for pT7- Hin dIII- ccdB to 57% for pT7- Hin dIII-dead- ccdB . ( d ) Image of the mixture of GFP + and GFP − resulting from the assembly (100 µl outgrowth medium spread). The red arrow points at a cluster of GFP − colonies representing un-digested vector that would have to be screened for in a non-model assembly.

    Techniques Used: Purification, Plasmid Preparation, Sequencing, Clone Assay, Derivative Assay

    3) Product Images from "FabV/Triclosan Is an Antibiotic-Free and Cost-Effective Selection System for Efficient Maintenance of High and Medium -Copy Number Plasmids in Escherichia coli"

    Article Title: FabV/Triclosan Is an Antibiotic-Free and Cost-Effective Selection System for Efficient Maintenance of High and Medium -Copy Number Plasmids in Escherichia coli

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0129547

    Plasmid DNA yield. E . coli DH5α and JM109 were transformed with high-copy number pUC19-Bla, pUC19-FabV, pUC19-FabI, and medium-copy number pSA-Hp24-Bla, pSA-Hp24-FabV, pBR322-FabI plasmids and selected on LB agar plates containing 1μM Triclosan (for FabV/FabI plasmids) and or 100μg mL -1 ampicillin (for Bla plasmids). Seed cultures were used to inoculated 5mL LB broth in 50mL flasks and cultured for another 18 hours at 37 or 30°C while shaking at 250rpm. Cell density was then measured by absorbing the diluted samples at 600nm and normalized to 2 OD600. One mL of the normalized culture was then used to extract the plasmid following supplied protocol. The quantity of the extracted plasmid DNA was measured by fluorometry using Qubit Fluorometer and Qubit dsDNA BR Assay Kit (Invitrogen, life technology). Fig 4A shows plasmid DNA yield when bacteria were grown at 37°C, whereas Fig 4B shows the plasmid DNA yield from cultures incubated at 30°C. Error bars show standard deviations calculated from at-least six (6) independent experiments performed in triplicate.
    Figure Legend Snippet: Plasmid DNA yield. E . coli DH5α and JM109 were transformed with high-copy number pUC19-Bla, pUC19-FabV, pUC19-FabI, and medium-copy number pSA-Hp24-Bla, pSA-Hp24-FabV, pBR322-FabI plasmids and selected on LB agar plates containing 1μM Triclosan (for FabV/FabI plasmids) and or 100μg mL -1 ampicillin (for Bla plasmids). Seed cultures were used to inoculated 5mL LB broth in 50mL flasks and cultured for another 18 hours at 37 or 30°C while shaking at 250rpm. Cell density was then measured by absorbing the diluted samples at 600nm and normalized to 2 OD600. One mL of the normalized culture was then used to extract the plasmid following supplied protocol. The quantity of the extracted plasmid DNA was measured by fluorometry using Qubit Fluorometer and Qubit dsDNA BR Assay Kit (Invitrogen, life technology). Fig 4A shows plasmid DNA yield when bacteria were grown at 37°C, whereas Fig 4B shows the plasmid DNA yield from cultures incubated at 30°C. Error bars show standard deviations calculated from at-least six (6) independent experiments performed in triplicate.

    Techniques Used: Plasmid Preparation, Transformation Assay, Cell Culture, Incubation

    Morphology of transformed bacteria. E . coli DH5α and JM109 transformed with (A) high-copy number pUC19-Bla, pUC19-FabV, or pUC19-FabI plasmids or (B) medium–copy number pMXB-p24-Bla, pMXBp24-FabV, or pBR322-FabI plasmids were incubated at 30°C for 18 hours on plates with our without selection agent (Ampicillin or Triclosan) and photographed. No appreciable differences noted when the transformants were plated on Agar plates containing selection agent or not.
    Figure Legend Snippet: Morphology of transformed bacteria. E . coli DH5α and JM109 transformed with (A) high-copy number pUC19-Bla, pUC19-FabV, or pUC19-FabI plasmids or (B) medium–copy number pMXB-p24-Bla, pMXBp24-FabV, or pBR322-FabI plasmids were incubated at 30°C for 18 hours on plates with our without selection agent (Ampicillin or Triclosan) and photographed. No appreciable differences noted when the transformants were plated on Agar plates containing selection agent or not.

    Techniques Used: Transformation Assay, Incubation, Selection

    Bacterial growth characteristics. E . coli DH5α, JM109, and BL21(DE3) were transformed with FabV (pUC19-FabV and pSA-HP24-FabV), FabI (pUC19-FabI, pBR322-FabI), or Bla (pUC19–Bla, pSA-HP24-Bla)-plasmids, and the transformants were selected on LB agar plates. Seed cultures were used to inoculate 25mL LB broth in 250mL baffled flasks and cultures grown at 37, 30, and 22°C for up to 12 hours while shaking at 250rpm. Samples were collected at one hour interval and growth was measured by absorbing the diluted samples at 600nm and graphs plotted. Error bars show standard deviations calculated from at-least six (6) independent experiments performed in triplicate.
    Figure Legend Snippet: Bacterial growth characteristics. E . coli DH5α, JM109, and BL21(DE3) were transformed with FabV (pUC19-FabV and pSA-HP24-FabV), FabI (pUC19-FabI, pBR322-FabI), or Bla (pUC19–Bla, pSA-HP24-Bla)-plasmids, and the transformants were selected on LB agar plates. Seed cultures were used to inoculate 25mL LB broth in 250mL baffled flasks and cultures grown at 37, 30, and 22°C for up to 12 hours while shaking at 250rpm. Samples were collected at one hour interval and growth was measured by absorbing the diluted samples at 600nm and graphs plotted. Error bars show standard deviations calculated from at-least six (6) independent experiments performed in triplicate.

    Techniques Used: Transformation Assay

    Bacterial transformation efficiency. Chemically competent E . coli DH5α and JM109 cells were transformed with 100pg μL -1 of purified high-copy number pUC19-Bla, pUC19-FabV, pUC19-FabI, and medium-copy number pSA-Hp24-Bla, pSA-Hp24-FabV, pBR322-FabI plasmids and selected on LB agar plates containing 1μM Triclosan (for FabV/FabI plasmids) and or 100μg mL -1 ampicillin (for Bla plasmids) after 18 hours of incubation at 30°C and transformation efficiency calculated. Fig 5A and B show transformation efficiency of various plasmids in DH5α and JM109 respectively. Fig 5C and D show the effect of incubation time prior to plating the transformants on selection plates. Error bars show standard deviations calculated from at-least six (6) independent experiments performed in triplicate.
    Figure Legend Snippet: Bacterial transformation efficiency. Chemically competent E . coli DH5α and JM109 cells were transformed with 100pg μL -1 of purified high-copy number pUC19-Bla, pUC19-FabV, pUC19-FabI, and medium-copy number pSA-Hp24-Bla, pSA-Hp24-FabV, pBR322-FabI plasmids and selected on LB agar plates containing 1μM Triclosan (for FabV/FabI plasmids) and or 100μg mL -1 ampicillin (for Bla plasmids) after 18 hours of incubation at 30°C and transformation efficiency calculated. Fig 5A and B show transformation efficiency of various plasmids in DH5α and JM109 respectively. Fig 5C and D show the effect of incubation time prior to plating the transformants on selection plates. Error bars show standard deviations calculated from at-least six (6) independent experiments performed in triplicate.

    Techniques Used: Electroporation Bacterial Transformation, Transformation Assay, Purification, Incubation, Selection

    4) Product Images from "ZeBRα a universal, multi-fragment DNA-assembly-system with minimal hands-on time requirement"

    Article Title: ZeBRα a universal, multi-fragment DNA-assembly-system with minimal hands-on time requirement

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-39768-0

    Three or four fragments can efficiently be assembled with PPY-cell extracts, while iVEC/“transformation-cloning” with three fragments is markedly less efficient. ( a ) The assembly of four fragments in a single reaction reduces the number of successful assemblies by a factor of two to ten as compared to the three-fragment-assembly. Cell-extracts were prepared from autoinduced PPY cells and the assemblies were purified prior transformation. ( b ) Assembly of three fragments by iVEC/“transformation-cloning” with NEB 5-alpha resulted in roughly 250 recombinant colonies/µg transformed DNA. A significant number of colonies harboring plasmids with defective inserts (grey bar) and PCR-template carry-over (dotted bar) were present on the plates.
    Figure Legend Snippet: Three or four fragments can efficiently be assembled with PPY-cell extracts, while iVEC/“transformation-cloning” with three fragments is markedly less efficient. ( a ) The assembly of four fragments in a single reaction reduces the number of successful assemblies by a factor of two to ten as compared to the three-fragment-assembly. Cell-extracts were prepared from autoinduced PPY cells and the assemblies were purified prior transformation. ( b ) Assembly of three fragments by iVEC/“transformation-cloning” with NEB 5-alpha resulted in roughly 250 recombinant colonies/µg transformed DNA. A significant number of colonies harboring plasmids with defective inserts (grey bar) and PCR-template carry-over (dotted bar) were present on the plates.

    Techniques Used: Clone Assay, Purification, Transformation Assay, Recombinant, Polymerase Chain Reaction

    Strategy for SLiCE optimization and evaluation. ( a ) Flow chart of the optimization process for generating a recombinogenic E. coli lysate. The PPY strain is a DH10B-derivative used to prepare the recombinogenic cell lysate and expresses the coding sequences for Redαβγ. The extracts, derived from arabinose autoinduced PPY-cells, were compared to extracts made from non-induced PPY-cells. ( b ) Structure of the examined non-ionic detergents used to prepare the recombinogenic PPY-extracts, CHAPS, Sulfo-Betain (SB-12), n-Octyl-β-D-thioglucopyranosid (OTG), n-Octyl-β-D-glucopyranosid (OG) Dodecyl-β-D-maltosid (DDM). ( c ) PPY-extracts were tested for their recombination capacity by assembling three DNA fragments with overlapping ends, to generate a recombinant plasmid constitutively expressing a blue chromoprotein. To examine the effects detergents had on the transformation, samples were split after the assembly reaction. One part was transformed into NEB 5-alpha unpurified; the other fraction was purified by silica-column chromatography prior to transformation.
    Figure Legend Snippet: Strategy for SLiCE optimization and evaluation. ( a ) Flow chart of the optimization process for generating a recombinogenic E. coli lysate. The PPY strain is a DH10B-derivative used to prepare the recombinogenic cell lysate and expresses the coding sequences for Redαβγ. The extracts, derived from arabinose autoinduced PPY-cells, were compared to extracts made from non-induced PPY-cells. ( b ) Structure of the examined non-ionic detergents used to prepare the recombinogenic PPY-extracts, CHAPS, Sulfo-Betain (SB-12), n-Octyl-β-D-thioglucopyranosid (OTG), n-Octyl-β-D-glucopyranosid (OG) Dodecyl-β-D-maltosid (DDM). ( c ) PPY-extracts were tested for their recombination capacity by assembling three DNA fragments with overlapping ends, to generate a recombinant plasmid constitutively expressing a blue chromoprotein. To examine the effects detergents had on the transformation, samples were split after the assembly reaction. One part was transformed into NEB 5-alpha unpurified; the other fraction was purified by silica-column chromatography prior to transformation.

    Techniques Used: Flow Cytometry, Derivative Assay, Recombinant, Plasmid Preparation, Expressing, Transformation Assay, Purification, Column Chromatography

    Comparison of the influence of detergent, autoinduction, post-assembly purification and competency of used bacteria on DNA assembling efficiency. ( a ) In four of the five PPY lysis conditions induction of Redα had moderate effects. Five different detergents were tested on PPY-cells grown with either lactose (Redα un-induced) or arabinose (Redα induced). All assemblies were column-purified before transformation into NEB 5-alpha. Bars indicate standard error of three independent replicates of an assembly reaction in all following graphs. PPY recombinogenic capacity was assessed in three-fragment assemblies. ( b ) Column-purification of the three-fragment-assembly reactions led to markedly increased number of recombinant colonies for all tested detergents. In the case of CHAPS and SB-12, unpurified samples resulted in no colonies. The OTG derived PPY-extract resulted in the highest number of recombinant colonies without purification. All assemblies were arabinose-induced. ( c ) Chemical competency has profound influence on recombination efficiency. OTG prepared PPY-lysate was used in a three-fragment-assembly and transformed into commercial NEB 5-alpha competent E. coli (1 × 10 9 cfu/µg pUC DNA) or the same strain prepared by the Inoue-method 21 (2.3 × 10 6 cfu/µg DNA). ( d ) For convenient readout of the potency of the PPY-extracts PCR-fragments used for the three- and four-way assembly reactions consisted of a blue chromoprotein coding ORF, a kanamycin resistance gene an origin-of-replication (on one fragment for the three-fragment assembly) and a bacterial basal-promoter-fragment. Only successful recombinants could produce blue colonies on kanamycin plates. The PCR-fragments to be assembled had overlapping bases that summed up to about 15 bp overlapping ends.
    Figure Legend Snippet: Comparison of the influence of detergent, autoinduction, post-assembly purification and competency of used bacteria on DNA assembling efficiency. ( a ) In four of the five PPY lysis conditions induction of Redα had moderate effects. Five different detergents were tested on PPY-cells grown with either lactose (Redα un-induced) or arabinose (Redα induced). All assemblies were column-purified before transformation into NEB 5-alpha. Bars indicate standard error of three independent replicates of an assembly reaction in all following graphs. PPY recombinogenic capacity was assessed in three-fragment assemblies. ( b ) Column-purification of the three-fragment-assembly reactions led to markedly increased number of recombinant colonies for all tested detergents. In the case of CHAPS and SB-12, unpurified samples resulted in no colonies. The OTG derived PPY-extract resulted in the highest number of recombinant colonies without purification. All assemblies were arabinose-induced. ( c ) Chemical competency has profound influence on recombination efficiency. OTG prepared PPY-lysate was used in a three-fragment-assembly and transformed into commercial NEB 5-alpha competent E. coli (1 × 10 9 cfu/µg pUC DNA) or the same strain prepared by the Inoue-method 21 (2.3 × 10 6 cfu/µg DNA). ( d ) For convenient readout of the potency of the PPY-extracts PCR-fragments used for the three- and four-way assembly reactions consisted of a blue chromoprotein coding ORF, a kanamycin resistance gene an origin-of-replication (on one fragment for the three-fragment assembly) and a bacterial basal-promoter-fragment. Only successful recombinants could produce blue colonies on kanamycin plates. The PCR-fragments to be assembled had overlapping bases that summed up to about 15 bp overlapping ends.

    Techniques Used: Purification, Lysis, Transformation Assay, Recombinant, Derivative Assay, Polymerase Chain Reaction

    The recombinogenic capacity of OTG extracts from autoinduced PPY and NEB 5-alpha extracts are equivalent or better than PPY-extracts generated by the original protocol. ( a ) Plasmid map of pT7- Hin dIII- ccdB used to assess three-fragment ZeBRα assemblies. Two Hin dIII and two Bsa I sites flank the toxic-placeholder- ccdB , allowing linearization and removal of ccdB . Unique sites are available on either side of ccdB . Chloramphenicol-acetyl-transferase coding gene ( CmR ), is part of the placeholder cassette and prevents ccdB -loss during plasmid propagation. The hatched region encompasses the fragment removed during cloning. ( b ) Map of the vector pT7-GFP antisense resulting from the three-fragment test-assembly of the pT7- Hin dIII- ccdB as recipient for a GFP-ORF and a bacterial promoter containing PCR-fragment, to evaluate the efficacy of the ZeBRα-procedure. Criss-cross lines mark the fusion-sites of the assembled fragments. ( c ) Comparison of the recombination capacity of extracts prepared with OTG or CelLyticB TM from manually induced and autoinduced (denoted as “auto” in the column) PPY-cells and NEB 5-alpha. The iVEC/“transformation-cloning” of the respective fragments is shown as last column. ( d ) Green fluorescent NEB 5-alpha colonies harboring the constitutively GFP-expressing vector pT7-GFP antisense.
    Figure Legend Snippet: The recombinogenic capacity of OTG extracts from autoinduced PPY and NEB 5-alpha extracts are equivalent or better than PPY-extracts generated by the original protocol. ( a ) Plasmid map of pT7- Hin dIII- ccdB used to assess three-fragment ZeBRα assemblies. Two Hin dIII and two Bsa I sites flank the toxic-placeholder- ccdB , allowing linearization and removal of ccdB . Unique sites are available on either side of ccdB . Chloramphenicol-acetyl-transferase coding gene ( CmR ), is part of the placeholder cassette and prevents ccdB -loss during plasmid propagation. The hatched region encompasses the fragment removed during cloning. ( b ) Map of the vector pT7-GFP antisense resulting from the three-fragment test-assembly of the pT7- Hin dIII- ccdB as recipient for a GFP-ORF and a bacterial promoter containing PCR-fragment, to evaluate the efficacy of the ZeBRα-procedure. Criss-cross lines mark the fusion-sites of the assembled fragments. ( c ) Comparison of the recombination capacity of extracts prepared with OTG or CelLyticB TM from manually induced and autoinduced (denoted as “auto” in the column) PPY-cells and NEB 5-alpha. The iVEC/“transformation-cloning” of the respective fragments is shown as last column. ( d ) Green fluorescent NEB 5-alpha colonies harboring the constitutively GFP-expressing vector pT7-GFP antisense.

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

    Rendering the CcdB in the placeholder non-toxic increases the number of GFP – background colonies markedly, showing that ccdB is an essential element if working with non-gel purified vector. ( a ) The pT7- Hin dIII-dead- ccdB differs by a four base-pair deletion in the ccdB -coding sequence from it’s predecessor pT7- Hin dIII- ccdB . The dotted lines encompass the region removed during cloning. ( b ) Sequence alignment of the region encompassing the small deletion in the ccdB -ORF, in pT7- Hin dIII-dead- ccdB compared to the region in pT7- Hin dIII- ccdB and the resulting frameshift rendering the ΔCcdB non-toxic for E. coli NEB 5-alpha. The numbers denote bases in the vector. ( c ) The three-fragment assembly as shown in Fig. 6c uses the pT7-HindIII-dead- ccdB vector and OTG-derived PPY-extract to assemble pT7-GFP antisense analogous to the assemblies shown in Fig. 5 . The percentage of GFP + colonies drops from nearly 100% for pT7- Hin dIII- ccdB to 57% for pT7- Hin dIII-dead- ccdB . ( d ) Image of the mixture of GFP + and GFP − resulting from the assembly (100 µl outgrowth medium spread). The red arrow points at a cluster of GFP − colonies representing un-digested vector that would have to be screened for in a non-model assembly.
    Figure Legend Snippet: Rendering the CcdB in the placeholder non-toxic increases the number of GFP – background colonies markedly, showing that ccdB is an essential element if working with non-gel purified vector. ( a ) The pT7- Hin dIII-dead- ccdB differs by a four base-pair deletion in the ccdB -coding sequence from it’s predecessor pT7- Hin dIII- ccdB . The dotted lines encompass the region removed during cloning. ( b ) Sequence alignment of the region encompassing the small deletion in the ccdB -ORF, in pT7- Hin dIII-dead- ccdB compared to the region in pT7- Hin dIII- ccdB and the resulting frameshift rendering the ΔCcdB non-toxic for E. coli NEB 5-alpha. The numbers denote bases in the vector. ( c ) The three-fragment assembly as shown in Fig. 6c uses the pT7-HindIII-dead- ccdB vector and OTG-derived PPY-extract to assemble pT7-GFP antisense analogous to the assemblies shown in Fig. 5 . The percentage of GFP + colonies drops from nearly 100% for pT7- Hin dIII- ccdB to 57% for pT7- Hin dIII-dead- ccdB . ( d ) Image of the mixture of GFP + and GFP − resulting from the assembly (100 µl outgrowth medium spread). The red arrow points at a cluster of GFP − colonies representing un-digested vector that would have to be screened for in a non-model assembly.

    Techniques Used: Purification, Plasmid Preparation, Sequencing, Clone Assay, Derivative Assay

    5) Product Images from "ZeBRα a universal, multi-fragment DNA-assembly-system with minimal hands-on time requirement"

    Article Title: ZeBRα a universal, multi-fragment DNA-assembly-system with minimal hands-on time requirement

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-39768-0

    Three or four fragments can efficiently be assembled with PPY-cell extracts, while iVEC/“transformation-cloning” with three fragments is markedly less efficient. ( a ) The assembly of four fragments in a single reaction reduces the number of successful assemblies by a factor of two to ten as compared to the three-fragment-assembly. Cell-extracts were prepared from autoinduced PPY cells and the assemblies were purified prior transformation. ( b ) Assembly of three fragments by iVEC/“transformation-cloning” with NEB 5-alpha resulted in roughly 250 recombinant colonies/µg transformed DNA. A significant number of colonies harboring plasmids with defective inserts (grey bar) and PCR-template carry-over (dotted bar) were present on the plates.
    Figure Legend Snippet: Three or four fragments can efficiently be assembled with PPY-cell extracts, while iVEC/“transformation-cloning” with three fragments is markedly less efficient. ( a ) The assembly of four fragments in a single reaction reduces the number of successful assemblies by a factor of two to ten as compared to the three-fragment-assembly. Cell-extracts were prepared from autoinduced PPY cells and the assemblies were purified prior transformation. ( b ) Assembly of three fragments by iVEC/“transformation-cloning” with NEB 5-alpha resulted in roughly 250 recombinant colonies/µg transformed DNA. A significant number of colonies harboring plasmids with defective inserts (grey bar) and PCR-template carry-over (dotted bar) were present on the plates.

    Techniques Used: Clone Assay, Purification, Transformation Assay, Recombinant, Polymerase Chain Reaction

    Strategy for SLiCE optimization and evaluation. ( a ) Flow chart of the optimization process for generating a recombinogenic E. coli lysate. The PPY strain is a DH10B-derivative used to prepare the recombinogenic cell lysate and expresses the coding sequences for Redαβγ. The extracts, derived from arabinose autoinduced PPY-cells, were compared to extracts made from non-induced PPY-cells. ( b ) Structure of the examined non-ionic detergents used to prepare the recombinogenic PPY-extracts, CHAPS, Sulfo-Betain (SB-12), n-Octyl-β-D-thioglucopyranosid (OTG), n-Octyl-β-D-glucopyranosid (OG) Dodecyl-β-D-maltosid (DDM). ( c ) PPY-extracts were tested for their recombination capacity by assembling three DNA fragments with overlapping ends, to generate a recombinant plasmid constitutively expressing a blue chromoprotein. To examine the effects detergents had on the transformation, samples were split after the assembly reaction. One part was transformed into NEB 5-alpha unpurified; the other fraction was purified by silica-column chromatography prior to transformation.
    Figure Legend Snippet: Strategy for SLiCE optimization and evaluation. ( a ) Flow chart of the optimization process for generating a recombinogenic E. coli lysate. The PPY strain is a DH10B-derivative used to prepare the recombinogenic cell lysate and expresses the coding sequences for Redαβγ. The extracts, derived from arabinose autoinduced PPY-cells, were compared to extracts made from non-induced PPY-cells. ( b ) Structure of the examined non-ionic detergents used to prepare the recombinogenic PPY-extracts, CHAPS, Sulfo-Betain (SB-12), n-Octyl-β-D-thioglucopyranosid (OTG), n-Octyl-β-D-glucopyranosid (OG) Dodecyl-β-D-maltosid (DDM). ( c ) PPY-extracts were tested for their recombination capacity by assembling three DNA fragments with overlapping ends, to generate a recombinant plasmid constitutively expressing a blue chromoprotein. To examine the effects detergents had on the transformation, samples were split after the assembly reaction. One part was transformed into NEB 5-alpha unpurified; the other fraction was purified by silica-column chromatography prior to transformation.

    Techniques Used: Flow Cytometry, Derivative Assay, Recombinant, Plasmid Preparation, Expressing, Transformation Assay, Purification, Column Chromatography

    Comparison of the influence of detergent, autoinduction, post-assembly purification and competency of used bacteria on DNA assembling efficiency. ( a ) In four of the five PPY lysis conditions induction of Redα had moderate effects. Five different detergents were tested on PPY-cells grown with either lactose (Redα un-induced) or arabinose (Redα induced). All assemblies were column-purified before transformation into NEB 5-alpha. Bars indicate standard error of three independent replicates of an assembly reaction in all following graphs. PPY recombinogenic capacity was assessed in three-fragment assemblies. ( b ) Column-purification of the three-fragment-assembly reactions led to markedly increased number of recombinant colonies for all tested detergents. In the case of CHAPS and SB-12, unpurified samples resulted in no colonies. The OTG derived PPY-extract resulted in the highest number of recombinant colonies without purification. All assemblies were arabinose-induced. ( c ) Chemical competency has profound influence on recombination efficiency. OTG prepared PPY-lysate was used in a three-fragment-assembly and transformed into commercial NEB 5-alpha competent E. coli (1 × 10 9 cfu/µg pUC DNA) or the same strain prepared by the Inoue-method 21 (2.3 × 10 6 cfu/µg DNA). ( d ) For convenient readout of the potency of the PPY-extracts PCR-fragments used for the three- and four-way assembly reactions consisted of a blue chromoprotein coding ORF, a kanamycin resistance gene an origin-of-replication (on one fragment for the three-fragment assembly) and a bacterial basal-promoter-fragment. Only successful recombinants could produce blue colonies on kanamycin plates. The PCR-fragments to be assembled had overlapping bases that summed up to about 15 bp overlapping ends.
    Figure Legend Snippet: Comparison of the influence of detergent, autoinduction, post-assembly purification and competency of used bacteria on DNA assembling efficiency. ( a ) In four of the five PPY lysis conditions induction of Redα had moderate effects. Five different detergents were tested on PPY-cells grown with either lactose (Redα un-induced) or arabinose (Redα induced). All assemblies were column-purified before transformation into NEB 5-alpha. Bars indicate standard error of three independent replicates of an assembly reaction in all following graphs. PPY recombinogenic capacity was assessed in three-fragment assemblies. ( b ) Column-purification of the three-fragment-assembly reactions led to markedly increased number of recombinant colonies for all tested detergents. In the case of CHAPS and SB-12, unpurified samples resulted in no colonies. The OTG derived PPY-extract resulted in the highest number of recombinant colonies without purification. All assemblies were arabinose-induced. ( c ) Chemical competency has profound influence on recombination efficiency. OTG prepared PPY-lysate was used in a three-fragment-assembly and transformed into commercial NEB 5-alpha competent E. coli (1 × 10 9 cfu/µg pUC DNA) or the same strain prepared by the Inoue-method 21 (2.3 × 10 6 cfu/µg DNA). ( d ) For convenient readout of the potency of the PPY-extracts PCR-fragments used for the three- and four-way assembly reactions consisted of a blue chromoprotein coding ORF, a kanamycin resistance gene an origin-of-replication (on one fragment for the three-fragment assembly) and a bacterial basal-promoter-fragment. Only successful recombinants could produce blue colonies on kanamycin plates. The PCR-fragments to be assembled had overlapping bases that summed up to about 15 bp overlapping ends.

    Techniques Used: Purification, Lysis, Transformation Assay, Recombinant, Derivative Assay, Polymerase Chain Reaction

    The recombinogenic capacity of OTG extracts from autoinduced PPY and NEB 5-alpha extracts are equivalent or better than PPY-extracts generated by the original protocol. ( a ) Plasmid map of pT7- Hin dIII- ccdB used to assess three-fragment ZeBRα assemblies. Two Hin dIII and two Bsa I sites flank the toxic-placeholder- ccdB , allowing linearization and removal of ccdB . Unique sites are available on either side of ccdB . Chloramphenicol-acetyl-transferase coding gene ( CmR ), is part of the placeholder cassette and prevents ccdB -loss during plasmid propagation. The hatched region encompasses the fragment removed during cloning. ( b ) Map of the vector pT7-GFP antisense resulting from the three-fragment test-assembly of the pT7- Hin dIII- ccdB as recipient for a GFP-ORF and a bacterial promoter containing PCR-fragment, to evaluate the efficacy of the ZeBRα-procedure. Criss-cross lines mark the fusion-sites of the assembled fragments. ( c ) Comparison of the recombination capacity of extracts prepared with OTG or CelLyticB TM from manually induced and autoinduced (denoted as “auto” in the column) PPY-cells and NEB 5-alpha. The iVEC/“transformation-cloning” of the respective fragments is shown as last column. ( d ) Green fluorescent NEB 5-alpha colonies harboring the constitutively GFP-expressing vector pT7-GFP antisense.
    Figure Legend Snippet: The recombinogenic capacity of OTG extracts from autoinduced PPY and NEB 5-alpha extracts are equivalent or better than PPY-extracts generated by the original protocol. ( a ) Plasmid map of pT7- Hin dIII- ccdB used to assess three-fragment ZeBRα assemblies. Two Hin dIII and two Bsa I sites flank the toxic-placeholder- ccdB , allowing linearization and removal of ccdB . Unique sites are available on either side of ccdB . Chloramphenicol-acetyl-transferase coding gene ( CmR ), is part of the placeholder cassette and prevents ccdB -loss during plasmid propagation. The hatched region encompasses the fragment removed during cloning. ( b ) Map of the vector pT7-GFP antisense resulting from the three-fragment test-assembly of the pT7- Hin dIII- ccdB as recipient for a GFP-ORF and a bacterial promoter containing PCR-fragment, to evaluate the efficacy of the ZeBRα-procedure. Criss-cross lines mark the fusion-sites of the assembled fragments. ( c ) Comparison of the recombination capacity of extracts prepared with OTG or CelLyticB TM from manually induced and autoinduced (denoted as “auto” in the column) PPY-cells and NEB 5-alpha. The iVEC/“transformation-cloning” of the respective fragments is shown as last column. ( d ) Green fluorescent NEB 5-alpha colonies harboring the constitutively GFP-expressing vector pT7-GFP antisense.

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

    Rendering the CcdB in the placeholder non-toxic increases the number of GFP – background colonies markedly, showing that ccdB is an essential element if working with non-gel purified vector. ( a ) The pT7- Hin dIII-dead- ccdB differs by a four base-pair deletion in the ccdB -coding sequence from it’s predecessor pT7- Hin dIII- ccdB . The dotted lines encompass the region removed during cloning. ( b ) Sequence alignment of the region encompassing the small deletion in the ccdB -ORF, in pT7- Hin dIII-dead- ccdB compared to the region in pT7- Hin dIII- ccdB and the resulting frameshift rendering the ΔCcdB non-toxic for E. coli NEB 5-alpha. The numbers denote bases in the vector. ( c ) The three-fragment assembly as shown in Fig. 6c uses the pT7-HindIII-dead- ccdB vector and OTG-derived PPY-extract to assemble pT7-GFP antisense analogous to the assemblies shown in Fig. 5 . The percentage of GFP + colonies drops from nearly 100% for pT7- Hin dIII- ccdB to 57% for pT7- Hin dIII-dead- ccdB . ( d ) Image of the mixture of GFP + and GFP − resulting from the assembly (100 µl outgrowth medium spread). The red arrow points at a cluster of GFP − colonies representing un-digested vector that would have to be screened for in a non-model assembly.
    Figure Legend Snippet: Rendering the CcdB in the placeholder non-toxic increases the number of GFP – background colonies markedly, showing that ccdB is an essential element if working with non-gel purified vector. ( a ) The pT7- Hin dIII-dead- ccdB differs by a four base-pair deletion in the ccdB -coding sequence from it’s predecessor pT7- Hin dIII- ccdB . The dotted lines encompass the region removed during cloning. ( b ) Sequence alignment of the region encompassing the small deletion in the ccdB -ORF, in pT7- Hin dIII-dead- ccdB compared to the region in pT7- Hin dIII- ccdB and the resulting frameshift rendering the ΔCcdB non-toxic for E. coli NEB 5-alpha. The numbers denote bases in the vector. ( c ) The three-fragment assembly as shown in Fig. 6c uses the pT7-HindIII-dead- ccdB vector and OTG-derived PPY-extract to assemble pT7-GFP antisense analogous to the assemblies shown in Fig. 5 . The percentage of GFP + colonies drops from nearly 100% for pT7- Hin dIII- ccdB to 57% for pT7- Hin dIII-dead- ccdB . ( d ) Image of the mixture of GFP + and GFP − resulting from the assembly (100 µl outgrowth medium spread). The red arrow points at a cluster of GFP − colonies representing un-digested vector that would have to be screened for in a non-model assembly.

    Techniques Used: Purification, Plasmid Preparation, Sequencing, Clone Assay, Derivative Assay

    6) Product Images from "FabV/Triclosan Is an Antibiotic-Free and Cost-Effective Selection System for Efficient Maintenance of High and Medium -Copy Number Plasmids in Escherichia coli"

    Article Title: FabV/Triclosan Is an Antibiotic-Free and Cost-Effective Selection System for Efficient Maintenance of High and Medium -Copy Number Plasmids in Escherichia coli

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0129547

    Plasmid DNA yield. E . coli DH5α and JM109 were transformed with high-copy number pUC19-Bla, pUC19-FabV, pUC19-FabI, and medium-copy number pSA-Hp24-Bla, pSA-Hp24-FabV, pBR322-FabI plasmids and selected on LB agar plates containing 1μM Triclosan (for FabV/FabI plasmids) and or 100μg mL -1 ampicillin (for Bla plasmids). Seed cultures were used to inoculated 5mL LB broth in 50mL flasks and cultured for another 18 hours at 37 or 30°C while shaking at 250rpm. Cell density was then measured by absorbing the diluted samples at 600nm and normalized to 2 OD600. One mL of the normalized culture was then used to extract the plasmid following supplied protocol. The quantity of the extracted plasmid DNA was measured by fluorometry using Qubit Fluorometer and Qubit dsDNA BR Assay Kit (Invitrogen, life technology). Fig 4A shows plasmid DNA yield when bacteria were grown at 37°C, whereas Fig 4B shows the plasmid DNA yield from cultures incubated at 30°C. Error bars show standard deviations calculated from at-least six (6) independent experiments performed in triplicate.
    Figure Legend Snippet: Plasmid DNA yield. E . coli DH5α and JM109 were transformed with high-copy number pUC19-Bla, pUC19-FabV, pUC19-FabI, and medium-copy number pSA-Hp24-Bla, pSA-Hp24-FabV, pBR322-FabI plasmids and selected on LB agar plates containing 1μM Triclosan (for FabV/FabI plasmids) and or 100μg mL -1 ampicillin (for Bla plasmids). Seed cultures were used to inoculated 5mL LB broth in 50mL flasks and cultured for another 18 hours at 37 or 30°C while shaking at 250rpm. Cell density was then measured by absorbing the diluted samples at 600nm and normalized to 2 OD600. One mL of the normalized culture was then used to extract the plasmid following supplied protocol. The quantity of the extracted plasmid DNA was measured by fluorometry using Qubit Fluorometer and Qubit dsDNA BR Assay Kit (Invitrogen, life technology). Fig 4A shows plasmid DNA yield when bacteria were grown at 37°C, whereas Fig 4B shows the plasmid DNA yield from cultures incubated at 30°C. Error bars show standard deviations calculated from at-least six (6) independent experiments performed in triplicate.

    Techniques Used: Plasmid Preparation, Transformation Assay, Cell Culture, Incubation

    Morphology of transformed bacteria. E . coli DH5α and JM109 transformed with (A) high-copy number pUC19-Bla, pUC19-FabV, or pUC19-FabI plasmids or (B) medium–copy number pMXB-p24-Bla, pMXBp24-FabV, or pBR322-FabI plasmids were incubated at 30°C for 18 hours on plates with our without selection agent (Ampicillin or Triclosan) and photographed. No appreciable differences noted when the transformants were plated on Agar plates containing selection agent or not.
    Figure Legend Snippet: Morphology of transformed bacteria. E . coli DH5α and JM109 transformed with (A) high-copy number pUC19-Bla, pUC19-FabV, or pUC19-FabI plasmids or (B) medium–copy number pMXB-p24-Bla, pMXBp24-FabV, or pBR322-FabI plasmids were incubated at 30°C for 18 hours on plates with our without selection agent (Ampicillin or Triclosan) and photographed. No appreciable differences noted when the transformants were plated on Agar plates containing selection agent or not.

    Techniques Used: Transformation Assay, Incubation, Selection

    Bacterial growth characteristics. E . coli DH5α, JM109, and BL21(DE3) were transformed with FabV (pUC19-FabV and pSA-HP24-FabV), FabI (pUC19-FabI, pBR322-FabI), or Bla (pUC19–Bla, pSA-HP24-Bla)-plasmids, and the transformants were selected on LB agar plates. Seed cultures were used to inoculate 25mL LB broth in 250mL baffled flasks and cultures grown at 37, 30, and 22°C for up to 12 hours while shaking at 250rpm. Samples were collected at one hour interval and growth was measured by absorbing the diluted samples at 600nm and graphs plotted. Error bars show standard deviations calculated from at-least six (6) independent experiments performed in triplicate.
    Figure Legend Snippet: Bacterial growth characteristics. E . coli DH5α, JM109, and BL21(DE3) were transformed with FabV (pUC19-FabV and pSA-HP24-FabV), FabI (pUC19-FabI, pBR322-FabI), or Bla (pUC19–Bla, pSA-HP24-Bla)-plasmids, and the transformants were selected on LB agar plates. Seed cultures were used to inoculate 25mL LB broth in 250mL baffled flasks and cultures grown at 37, 30, and 22°C for up to 12 hours while shaking at 250rpm. Samples were collected at one hour interval and growth was measured by absorbing the diluted samples at 600nm and graphs plotted. Error bars show standard deviations calculated from at-least six (6) independent experiments performed in triplicate.

    Techniques Used: Transformation Assay

    Bacterial transformation efficiency. Chemically competent E . coli DH5α and JM109 cells were transformed with 100pg μL -1 of purified high-copy number pUC19-Bla, pUC19-FabV, pUC19-FabI, and medium-copy number pSA-Hp24-Bla, pSA-Hp24-FabV, pBR322-FabI plasmids and selected on LB agar plates containing 1μM Triclosan (for FabV/FabI plasmids) and or 100μg mL -1 ampicillin (for Bla plasmids) after 18 hours of incubation at 30°C and transformation efficiency calculated. Fig 5A and B show transformation efficiency of various plasmids in DH5α and JM109 respectively. Fig 5C and D show the effect of incubation time prior to plating the transformants on selection plates. Error bars show standard deviations calculated from at-least six (6) independent experiments performed in triplicate.
    Figure Legend Snippet: Bacterial transformation efficiency. Chemically competent E . coli DH5α and JM109 cells were transformed with 100pg μL -1 of purified high-copy number pUC19-Bla, pUC19-FabV, pUC19-FabI, and medium-copy number pSA-Hp24-Bla, pSA-Hp24-FabV, pBR322-FabI plasmids and selected on LB agar plates containing 1μM Triclosan (for FabV/FabI plasmids) and or 100μg mL -1 ampicillin (for Bla plasmids) after 18 hours of incubation at 30°C and transformation efficiency calculated. Fig 5A and B show transformation efficiency of various plasmids in DH5α and JM109 respectively. Fig 5C and D show the effect of incubation time prior to plating the transformants on selection plates. Error bars show standard deviations calculated from at-least six (6) independent experiments performed in triplicate.

    Techniques Used: Electroporation Bacterial Transformation, Transformation Assay, Purification, Incubation, Selection

    7) Product Images from "ZeBRα a universal, multi-fragment DNA-assembly-system with minimal hands-on time requirement"

    Article Title: ZeBRα a universal, multi-fragment DNA-assembly-system with minimal hands-on time requirement

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-39768-0

    Three or four fragments can efficiently be assembled with PPY-cell extracts, while iVEC/“transformation-cloning” with three fragments is markedly less efficient. ( a ) The assembly of four fragments in a single reaction reduces the number of successful assemblies by a factor of two to ten as compared to the three-fragment-assembly. Cell-extracts were prepared from autoinduced PPY cells and the assemblies were purified prior transformation. ( b ) Assembly of three fragments by iVEC/“transformation-cloning” with NEB 5-alpha resulted in roughly 250 recombinant colonies/µg transformed DNA. A significant number of colonies harboring plasmids with defective inserts (grey bar) and PCR-template carry-over (dotted bar) were present on the plates.
    Figure Legend Snippet: Three or four fragments can efficiently be assembled with PPY-cell extracts, while iVEC/“transformation-cloning” with three fragments is markedly less efficient. ( a ) The assembly of four fragments in a single reaction reduces the number of successful assemblies by a factor of two to ten as compared to the three-fragment-assembly. Cell-extracts were prepared from autoinduced PPY cells and the assemblies were purified prior transformation. ( b ) Assembly of three fragments by iVEC/“transformation-cloning” with NEB 5-alpha resulted in roughly 250 recombinant colonies/µg transformed DNA. A significant number of colonies harboring plasmids with defective inserts (grey bar) and PCR-template carry-over (dotted bar) were present on the plates.

    Techniques Used: Clone Assay, Purification, Transformation Assay, Recombinant, Polymerase Chain Reaction

    Strategy for SLiCE optimization and evaluation. ( a ) Flow chart of the optimization process for generating a recombinogenic E. coli lysate. The PPY strain is a DH10B-derivative used to prepare the recombinogenic cell lysate and expresses the coding sequences for Redαβγ. The extracts, derived from arabinose autoinduced PPY-cells, were compared to extracts made from non-induced PPY-cells. ( b ) Structure of the examined non-ionic detergents used to prepare the recombinogenic PPY-extracts, CHAPS, Sulfo-Betain (SB-12), n-Octyl-β-D-thioglucopyranosid (OTG), n-Octyl-β-D-glucopyranosid (OG) Dodecyl-β-D-maltosid (DDM). ( c ) PPY-extracts were tested for their recombination capacity by assembling three DNA fragments with overlapping ends, to generate a recombinant plasmid constitutively expressing a blue chromoprotein. To examine the effects detergents had on the transformation, samples were split after the assembly reaction. One part was transformed into NEB 5-alpha unpurified; the other fraction was purified by silica-column chromatography prior to transformation.
    Figure Legend Snippet: Strategy for SLiCE optimization and evaluation. ( a ) Flow chart of the optimization process for generating a recombinogenic E. coli lysate. The PPY strain is a DH10B-derivative used to prepare the recombinogenic cell lysate and expresses the coding sequences for Redαβγ. The extracts, derived from arabinose autoinduced PPY-cells, were compared to extracts made from non-induced PPY-cells. ( b ) Structure of the examined non-ionic detergents used to prepare the recombinogenic PPY-extracts, CHAPS, Sulfo-Betain (SB-12), n-Octyl-β-D-thioglucopyranosid (OTG), n-Octyl-β-D-glucopyranosid (OG) Dodecyl-β-D-maltosid (DDM). ( c ) PPY-extracts were tested for their recombination capacity by assembling three DNA fragments with overlapping ends, to generate a recombinant plasmid constitutively expressing a blue chromoprotein. To examine the effects detergents had on the transformation, samples were split after the assembly reaction. One part was transformed into NEB 5-alpha unpurified; the other fraction was purified by silica-column chromatography prior to transformation.

    Techniques Used: Flow Cytometry, Derivative Assay, Recombinant, Plasmid Preparation, Expressing, Transformation Assay, Purification, Column Chromatography

    Comparison of the influence of detergent, autoinduction, post-assembly purification and competency of used bacteria on DNA assembling efficiency. ( a ) In four of the five PPY lysis conditions induction of Redα had moderate effects. Five different detergents were tested on PPY-cells grown with either lactose (Redα un-induced) or arabinose (Redα induced). All assemblies were column-purified before transformation into NEB 5-alpha. Bars indicate standard error of three independent replicates of an assembly reaction in all following graphs. PPY recombinogenic capacity was assessed in three-fragment assemblies. ( b ) Column-purification of the three-fragment-assembly reactions led to markedly increased number of recombinant colonies for all tested detergents. In the case of CHAPS and SB-12, unpurified samples resulted in no colonies. The OTG derived PPY-extract resulted in the highest number of recombinant colonies without purification. All assemblies were arabinose-induced. ( c ) Chemical competency has profound influence on recombination efficiency. OTG prepared PPY-lysate was used in a three-fragment-assembly and transformed into commercial NEB 5-alpha competent E. coli (1 × 10 9 cfu/µg pUC DNA) or the same strain prepared by the Inoue-method 21 (2.3 × 10 6 cfu/µg DNA). ( d ) For convenient readout of the potency of the PPY-extracts PCR-fragments used for the three- and four-way assembly reactions consisted of a blue chromoprotein coding ORF, a kanamycin resistance gene an origin-of-replication (on one fragment for the three-fragment assembly) and a bacterial basal-promoter-fragment. Only successful recombinants could produce blue colonies on kanamycin plates. The PCR-fragments to be assembled had overlapping bases that summed up to about 15 bp overlapping ends.
    Figure Legend Snippet: Comparison of the influence of detergent, autoinduction, post-assembly purification and competency of used bacteria on DNA assembling efficiency. ( a ) In four of the five PPY lysis conditions induction of Redα had moderate effects. Five different detergents were tested on PPY-cells grown with either lactose (Redα un-induced) or arabinose (Redα induced). All assemblies were column-purified before transformation into NEB 5-alpha. Bars indicate standard error of three independent replicates of an assembly reaction in all following graphs. PPY recombinogenic capacity was assessed in three-fragment assemblies. ( b ) Column-purification of the three-fragment-assembly reactions led to markedly increased number of recombinant colonies for all tested detergents. In the case of CHAPS and SB-12, unpurified samples resulted in no colonies. The OTG derived PPY-extract resulted in the highest number of recombinant colonies without purification. All assemblies were arabinose-induced. ( c ) Chemical competency has profound influence on recombination efficiency. OTG prepared PPY-lysate was used in a three-fragment-assembly and transformed into commercial NEB 5-alpha competent E. coli (1 × 10 9 cfu/µg pUC DNA) or the same strain prepared by the Inoue-method 21 (2.3 × 10 6 cfu/µg DNA). ( d ) For convenient readout of the potency of the PPY-extracts PCR-fragments used for the three- and four-way assembly reactions consisted of a blue chromoprotein coding ORF, a kanamycin resistance gene an origin-of-replication (on one fragment for the three-fragment assembly) and a bacterial basal-promoter-fragment. Only successful recombinants could produce blue colonies on kanamycin plates. The PCR-fragments to be assembled had overlapping bases that summed up to about 15 bp overlapping ends.

    Techniques Used: Purification, Lysis, Transformation Assay, Recombinant, Derivative Assay, Polymerase Chain Reaction

    The recombinogenic capacity of OTG extracts from autoinduced PPY and NEB 5-alpha extracts are equivalent or better than PPY-extracts generated by the original protocol. ( a ) Plasmid map of pT7- Hin dIII- ccdB used to assess three-fragment ZeBRα assemblies. Two Hin dIII and two Bsa I sites flank the toxic-placeholder- ccdB , allowing linearization and removal of ccdB . Unique sites are available on either side of ccdB . Chloramphenicol-acetyl-transferase coding gene ( CmR ), is part of the placeholder cassette and prevents ccdB -loss during plasmid propagation. The hatched region encompasses the fragment removed during cloning. ( b ) Map of the vector pT7-GFP antisense resulting from the three-fragment test-assembly of the pT7- Hin dIII- ccdB as recipient for a GFP-ORF and a bacterial promoter containing PCR-fragment, to evaluate the efficacy of the ZeBRα-procedure. Criss-cross lines mark the fusion-sites of the assembled fragments. ( c ) Comparison of the recombination capacity of extracts prepared with OTG or CelLyticB TM from manually induced and autoinduced (denoted as “auto” in the column) PPY-cells and NEB 5-alpha. The iVEC/“transformation-cloning” of the respective fragments is shown as last column. ( d ) Green fluorescent NEB 5-alpha colonies harboring the constitutively GFP-expressing vector pT7-GFP antisense.
    Figure Legend Snippet: The recombinogenic capacity of OTG extracts from autoinduced PPY and NEB 5-alpha extracts are equivalent or better than PPY-extracts generated by the original protocol. ( a ) Plasmid map of pT7- Hin dIII- ccdB used to assess three-fragment ZeBRα assemblies. Two Hin dIII and two Bsa I sites flank the toxic-placeholder- ccdB , allowing linearization and removal of ccdB . Unique sites are available on either side of ccdB . Chloramphenicol-acetyl-transferase coding gene ( CmR ), is part of the placeholder cassette and prevents ccdB -loss during plasmid propagation. The hatched region encompasses the fragment removed during cloning. ( b ) Map of the vector pT7-GFP antisense resulting from the three-fragment test-assembly of the pT7- Hin dIII- ccdB as recipient for a GFP-ORF and a bacterial promoter containing PCR-fragment, to evaluate the efficacy of the ZeBRα-procedure. Criss-cross lines mark the fusion-sites of the assembled fragments. ( c ) Comparison of the recombination capacity of extracts prepared with OTG or CelLyticB TM from manually induced and autoinduced (denoted as “auto” in the column) PPY-cells and NEB 5-alpha. The iVEC/“transformation-cloning” of the respective fragments is shown as last column. ( d ) Green fluorescent NEB 5-alpha colonies harboring the constitutively GFP-expressing vector pT7-GFP antisense.

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

    Rendering the CcdB in the placeholder non-toxic increases the number of GFP – background colonies markedly, showing that ccdB is an essential element if working with non-gel purified vector. ( a ) The pT7- Hin dIII-dead- ccdB differs by a four base-pair deletion in the ccdB -coding sequence from it’s predecessor pT7- Hin dIII- ccdB . The dotted lines encompass the region removed during cloning. ( b ) Sequence alignment of the region encompassing the small deletion in the ccdB -ORF, in pT7- Hin dIII-dead- ccdB compared to the region in pT7- Hin dIII- ccdB and the resulting frameshift rendering the ΔCcdB non-toxic for E. coli NEB 5-alpha. The numbers denote bases in the vector. ( c ) The three-fragment assembly as shown in Fig. 6c uses the pT7-HindIII-dead- ccdB vector and OTG-derived PPY-extract to assemble pT7-GFP antisense analogous to the assemblies shown in Fig. 5 . The percentage of GFP + colonies drops from nearly 100% for pT7- Hin dIII- ccdB to 57% for pT7- Hin dIII-dead- ccdB . ( d ) Image of the mixture of GFP + and GFP − resulting from the assembly (100 µl outgrowth medium spread). The red arrow points at a cluster of GFP − colonies representing un-digested vector that would have to be screened for in a non-model assembly.
    Figure Legend Snippet: Rendering the CcdB in the placeholder non-toxic increases the number of GFP – background colonies markedly, showing that ccdB is an essential element if working with non-gel purified vector. ( a ) The pT7- Hin dIII-dead- ccdB differs by a four base-pair deletion in the ccdB -coding sequence from it’s predecessor pT7- Hin dIII- ccdB . The dotted lines encompass the region removed during cloning. ( b ) Sequence alignment of the region encompassing the small deletion in the ccdB -ORF, in pT7- Hin dIII-dead- ccdB compared to the region in pT7- Hin dIII- ccdB and the resulting frameshift rendering the ΔCcdB non-toxic for E. coli NEB 5-alpha. The numbers denote bases in the vector. ( c ) The three-fragment assembly as shown in Fig. 6c uses the pT7-HindIII-dead- ccdB vector and OTG-derived PPY-extract to assemble pT7-GFP antisense analogous to the assemblies shown in Fig. 5 . The percentage of GFP + colonies drops from nearly 100% for pT7- Hin dIII- ccdB to 57% for pT7- Hin dIII-dead- ccdB . ( d ) Image of the mixture of GFP + and GFP − resulting from the assembly (100 µl outgrowth medium spread). The red arrow points at a cluster of GFP − colonies representing un-digested vector that would have to be screened for in a non-model assembly.

    Techniques Used: Purification, Plasmid Preparation, Sequencing, Clone Assay, Derivative Assay

    8) Product Images from "ZeBRα a universal, multi-fragment DNA-assembly-system with minimal hands-on time requirement"

    Article Title: ZeBRα a universal, multi-fragment DNA-assembly-system with minimal hands-on time requirement

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-39768-0

    Three or four fragments can efficiently be assembled with PPY-cell extracts, while iVEC/“transformation-cloning” with three fragments is markedly less efficient. ( a ) The assembly of four fragments in a single reaction reduces the number of successful assemblies by a factor of two to ten as compared to the three-fragment-assembly. Cell-extracts were prepared from autoinduced PPY cells and the assemblies were purified prior transformation. ( b ) Assembly of three fragments by iVEC/“transformation-cloning” with NEB 5-alpha resulted in roughly 250 recombinant colonies/µg transformed DNA. A significant number of colonies harboring plasmids with defective inserts (grey bar) and PCR-template carry-over (dotted bar) were present on the plates.
    Figure Legend Snippet: Three or four fragments can efficiently be assembled with PPY-cell extracts, while iVEC/“transformation-cloning” with three fragments is markedly less efficient. ( a ) The assembly of four fragments in a single reaction reduces the number of successful assemblies by a factor of two to ten as compared to the three-fragment-assembly. Cell-extracts were prepared from autoinduced PPY cells and the assemblies were purified prior transformation. ( b ) Assembly of three fragments by iVEC/“transformation-cloning” with NEB 5-alpha resulted in roughly 250 recombinant colonies/µg transformed DNA. A significant number of colonies harboring plasmids with defective inserts (grey bar) and PCR-template carry-over (dotted bar) were present on the plates.

    Techniques Used: Clone Assay, Purification, Transformation Assay, Recombinant, Polymerase Chain Reaction

    Strategy for SLiCE optimization and evaluation. ( a ) Flow chart of the optimization process for generating a recombinogenic E. coli lysate. The PPY strain is a DH10B-derivative used to prepare the recombinogenic cell lysate and expresses the coding sequences for Redαβγ. The extracts, derived from arabinose autoinduced PPY-cells, were compared to extracts made from non-induced PPY-cells. ( b ) Structure of the examined non-ionic detergents used to prepare the recombinogenic PPY-extracts, CHAPS, Sulfo-Betain (SB-12), n-Octyl-β-D-thioglucopyranosid (OTG), n-Octyl-β-D-glucopyranosid (OG) Dodecyl-β-D-maltosid (DDM). ( c ) PPY-extracts were tested for their recombination capacity by assembling three DNA fragments with overlapping ends, to generate a recombinant plasmid constitutively expressing a blue chromoprotein. To examine the effects detergents had on the transformation, samples were split after the assembly reaction. One part was transformed into NEB 5-alpha unpurified; the other fraction was purified by silica-column chromatography prior to transformation.
    Figure Legend Snippet: Strategy for SLiCE optimization and evaluation. ( a ) Flow chart of the optimization process for generating a recombinogenic E. coli lysate. The PPY strain is a DH10B-derivative used to prepare the recombinogenic cell lysate and expresses the coding sequences for Redαβγ. The extracts, derived from arabinose autoinduced PPY-cells, were compared to extracts made from non-induced PPY-cells. ( b ) Structure of the examined non-ionic detergents used to prepare the recombinogenic PPY-extracts, CHAPS, Sulfo-Betain (SB-12), n-Octyl-β-D-thioglucopyranosid (OTG), n-Octyl-β-D-glucopyranosid (OG) Dodecyl-β-D-maltosid (DDM). ( c ) PPY-extracts were tested for their recombination capacity by assembling three DNA fragments with overlapping ends, to generate a recombinant plasmid constitutively expressing a blue chromoprotein. To examine the effects detergents had on the transformation, samples were split after the assembly reaction. One part was transformed into NEB 5-alpha unpurified; the other fraction was purified by silica-column chromatography prior to transformation.

    Techniques Used: Flow Cytometry, Derivative Assay, Recombinant, Plasmid Preparation, Expressing, Transformation Assay, Purification, Column Chromatography

    Comparison of the influence of detergent, autoinduction, post-assembly purification and competency of used bacteria on DNA assembling efficiency. ( a ) In four of the five PPY lysis conditions induction of Redα had moderate effects. Five different detergents were tested on PPY-cells grown with either lactose (Redα un-induced) or arabinose (Redα induced). All assemblies were column-purified before transformation into NEB 5-alpha. Bars indicate standard error of three independent replicates of an assembly reaction in all following graphs. PPY recombinogenic capacity was assessed in three-fragment assemblies. ( b ) Column-purification of the three-fragment-assembly reactions led to markedly increased number of recombinant colonies for all tested detergents. In the case of CHAPS and SB-12, unpurified samples resulted in no colonies. The OTG derived PPY-extract resulted in the highest number of recombinant colonies without purification. All assemblies were arabinose-induced. ( c ) Chemical competency has profound influence on recombination efficiency. OTG prepared PPY-lysate was used in a three-fragment-assembly and transformed into commercial NEB 5-alpha competent E. coli (1 × 10 9 cfu/µg pUC DNA) or the same strain prepared by the Inoue-method 21 (2.3 × 10 6 cfu/µg DNA). ( d ) For convenient readout of the potency of the PPY-extracts PCR-fragments used for the three- and four-way assembly reactions consisted of a blue chromoprotein coding ORF, a kanamycin resistance gene an origin-of-replication (on one fragment for the three-fragment assembly) and a bacterial basal-promoter-fragment. Only successful recombinants could produce blue colonies on kanamycin plates. The PCR-fragments to be assembled had overlapping bases that summed up to about 15 bp overlapping ends.
    Figure Legend Snippet: Comparison of the influence of detergent, autoinduction, post-assembly purification and competency of used bacteria on DNA assembling efficiency. ( a ) In four of the five PPY lysis conditions induction of Redα had moderate effects. Five different detergents were tested on PPY-cells grown with either lactose (Redα un-induced) or arabinose (Redα induced). All assemblies were column-purified before transformation into NEB 5-alpha. Bars indicate standard error of three independent replicates of an assembly reaction in all following graphs. PPY recombinogenic capacity was assessed in three-fragment assemblies. ( b ) Column-purification of the three-fragment-assembly reactions led to markedly increased number of recombinant colonies for all tested detergents. In the case of CHAPS and SB-12, unpurified samples resulted in no colonies. The OTG derived PPY-extract resulted in the highest number of recombinant colonies without purification. All assemblies were arabinose-induced. ( c ) Chemical competency has profound influence on recombination efficiency. OTG prepared PPY-lysate was used in a three-fragment-assembly and transformed into commercial NEB 5-alpha competent E. coli (1 × 10 9 cfu/µg pUC DNA) or the same strain prepared by the Inoue-method 21 (2.3 × 10 6 cfu/µg DNA). ( d ) For convenient readout of the potency of the PPY-extracts PCR-fragments used for the three- and four-way assembly reactions consisted of a blue chromoprotein coding ORF, a kanamycin resistance gene an origin-of-replication (on one fragment for the three-fragment assembly) and a bacterial basal-promoter-fragment. Only successful recombinants could produce blue colonies on kanamycin plates. The PCR-fragments to be assembled had overlapping bases that summed up to about 15 bp overlapping ends.

    Techniques Used: Purification, Lysis, Transformation Assay, Recombinant, Derivative Assay, Polymerase Chain Reaction

    The recombinogenic capacity of OTG extracts from autoinduced PPY and NEB 5-alpha extracts are equivalent or better than PPY-extracts generated by the original protocol. ( a ) Plasmid map of pT7- Hin dIII- ccdB used to assess three-fragment ZeBRα assemblies. Two Hin dIII and two Bsa I sites flank the toxic-placeholder- ccdB , allowing linearization and removal of ccdB . Unique sites are available on either side of ccdB . Chloramphenicol-acetyl-transferase coding gene ( CmR ), is part of the placeholder cassette and prevents ccdB -loss during plasmid propagation. The hatched region encompasses the fragment removed during cloning. ( b ) Map of the vector pT7-GFP antisense resulting from the three-fragment test-assembly of the pT7- Hin dIII- ccdB as recipient for a GFP-ORF and a bacterial promoter containing PCR-fragment, to evaluate the efficacy of the ZeBRα-procedure. Criss-cross lines mark the fusion-sites of the assembled fragments. ( c ) Comparison of the recombination capacity of extracts prepared with OTG or CelLyticB TM from manually induced and autoinduced (denoted as “auto” in the column) PPY-cells and NEB 5-alpha. The iVEC/“transformation-cloning” of the respective fragments is shown as last column. ( d ) Green fluorescent NEB 5-alpha colonies harboring the constitutively GFP-expressing vector pT7-GFP antisense.
    Figure Legend Snippet: The recombinogenic capacity of OTG extracts from autoinduced PPY and NEB 5-alpha extracts are equivalent or better than PPY-extracts generated by the original protocol. ( a ) Plasmid map of pT7- Hin dIII- ccdB used to assess three-fragment ZeBRα assemblies. Two Hin dIII and two Bsa I sites flank the toxic-placeholder- ccdB , allowing linearization and removal of ccdB . Unique sites are available on either side of ccdB . Chloramphenicol-acetyl-transferase coding gene ( CmR ), is part of the placeholder cassette and prevents ccdB -loss during plasmid propagation. The hatched region encompasses the fragment removed during cloning. ( b ) Map of the vector pT7-GFP antisense resulting from the three-fragment test-assembly of the pT7- Hin dIII- ccdB as recipient for a GFP-ORF and a bacterial promoter containing PCR-fragment, to evaluate the efficacy of the ZeBRα-procedure. Criss-cross lines mark the fusion-sites of the assembled fragments. ( c ) Comparison of the recombination capacity of extracts prepared with OTG or CelLyticB TM from manually induced and autoinduced (denoted as “auto” in the column) PPY-cells and NEB 5-alpha. The iVEC/“transformation-cloning” of the respective fragments is shown as last column. ( d ) Green fluorescent NEB 5-alpha colonies harboring the constitutively GFP-expressing vector pT7-GFP antisense.

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

    Rendering the CcdB in the placeholder non-toxic increases the number of GFP – background colonies markedly, showing that ccdB is an essential element if working with non-gel purified vector. ( a ) The pT7- Hin dIII-dead- ccdB differs by a four base-pair deletion in the ccdB -coding sequence from it’s predecessor pT7- Hin dIII- ccdB . The dotted lines encompass the region removed during cloning. ( b ) Sequence alignment of the region encompassing the small deletion in the ccdB -ORF, in pT7- Hin dIII-dead- ccdB compared to the region in pT7- Hin dIII- ccdB and the resulting frameshift rendering the ΔCcdB non-toxic for E. coli NEB 5-alpha. The numbers denote bases in the vector. ( c ) The three-fragment assembly as shown in Fig. 6c uses the pT7-HindIII-dead- ccdB vector and OTG-derived PPY-extract to assemble pT7-GFP antisense analogous to the assemblies shown in Fig. 5 . The percentage of GFP + colonies drops from nearly 100% for pT7- Hin dIII- ccdB to 57% for pT7- Hin dIII-dead- ccdB . ( d ) Image of the mixture of GFP + and GFP − resulting from the assembly (100 µl outgrowth medium spread). The red arrow points at a cluster of GFP − colonies representing un-digested vector that would have to be screened for in a non-model assembly.
    Figure Legend Snippet: Rendering the CcdB in the placeholder non-toxic increases the number of GFP – background colonies markedly, showing that ccdB is an essential element if working with non-gel purified vector. ( a ) The pT7- Hin dIII-dead- ccdB differs by a four base-pair deletion in the ccdB -coding sequence from it’s predecessor pT7- Hin dIII- ccdB . The dotted lines encompass the region removed during cloning. ( b ) Sequence alignment of the region encompassing the small deletion in the ccdB -ORF, in pT7- Hin dIII-dead- ccdB compared to the region in pT7- Hin dIII- ccdB and the resulting frameshift rendering the ΔCcdB non-toxic for E. coli NEB 5-alpha. The numbers denote bases in the vector. ( c ) The three-fragment assembly as shown in Fig. 6c uses the pT7-HindIII-dead- ccdB vector and OTG-derived PPY-extract to assemble pT7-GFP antisense analogous to the assemblies shown in Fig. 5 . The percentage of GFP + colonies drops from nearly 100% for pT7- Hin dIII- ccdB to 57% for pT7- Hin dIII-dead- ccdB . ( d ) Image of the mixture of GFP + and GFP − resulting from the assembly (100 µl outgrowth medium spread). The red arrow points at a cluster of GFP − colonies representing un-digested vector that would have to be screened for in a non-model assembly.

    Techniques Used: Purification, Plasmid Preparation, Sequencing, Clone Assay, Derivative Assay

    9) Product Images from "ZeBRα a universal, multi-fragment DNA-assembly-system with minimal hands-on time requirement"

    Article Title: ZeBRα a universal, multi-fragment DNA-assembly-system with minimal hands-on time requirement

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-39768-0

    Three or four fragments can efficiently be assembled with PPY-cell extracts, while iVEC/“transformation-cloning” with three fragments is markedly less efficient. ( a ) The assembly of four fragments in a single reaction reduces the number of successful assemblies by a factor of two to ten as compared to the three-fragment-assembly. Cell-extracts were prepared from autoinduced PPY cells and the assemblies were purified prior transformation. ( b ) Assembly of three fragments by iVEC/“transformation-cloning” with NEB 5-alpha resulted in roughly 250 recombinant colonies/µg transformed DNA. A significant number of colonies harboring plasmids with defective inserts (grey bar) and PCR-template carry-over (dotted bar) were present on the plates.
    Figure Legend Snippet: Three or four fragments can efficiently be assembled with PPY-cell extracts, while iVEC/“transformation-cloning” with three fragments is markedly less efficient. ( a ) The assembly of four fragments in a single reaction reduces the number of successful assemblies by a factor of two to ten as compared to the three-fragment-assembly. Cell-extracts were prepared from autoinduced PPY cells and the assemblies were purified prior transformation. ( b ) Assembly of three fragments by iVEC/“transformation-cloning” with NEB 5-alpha resulted in roughly 250 recombinant colonies/µg transformed DNA. A significant number of colonies harboring plasmids with defective inserts (grey bar) and PCR-template carry-over (dotted bar) were present on the plates.

    Techniques Used: Clone Assay, Purification, Transformation Assay, Recombinant, Polymerase Chain Reaction

    Strategy for SLiCE optimization and evaluation. ( a ) Flow chart of the optimization process for generating a recombinogenic E. coli lysate. The PPY strain is a DH10B-derivative used to prepare the recombinogenic cell lysate and expresses the coding sequences for Redαβγ. The extracts, derived from arabinose autoinduced PPY-cells, were compared to extracts made from non-induced PPY-cells. ( b ) Structure of the examined non-ionic detergents used to prepare the recombinogenic PPY-extracts, CHAPS, Sulfo-Betain (SB-12), n-Octyl-β-D-thioglucopyranosid (OTG), n-Octyl-β-D-glucopyranosid (OG) Dodecyl-β-D-maltosid (DDM). ( c ) PPY-extracts were tested for their recombination capacity by assembling three DNA fragments with overlapping ends, to generate a recombinant plasmid constitutively expressing a blue chromoprotein. To examine the effects detergents had on the transformation, samples were split after the assembly reaction. One part was transformed into NEB 5-alpha unpurified; the other fraction was purified by silica-column chromatography prior to transformation.
    Figure Legend Snippet: Strategy for SLiCE optimization and evaluation. ( a ) Flow chart of the optimization process for generating a recombinogenic E. coli lysate. The PPY strain is a DH10B-derivative used to prepare the recombinogenic cell lysate and expresses the coding sequences for Redαβγ. The extracts, derived from arabinose autoinduced PPY-cells, were compared to extracts made from non-induced PPY-cells. ( b ) Structure of the examined non-ionic detergents used to prepare the recombinogenic PPY-extracts, CHAPS, Sulfo-Betain (SB-12), n-Octyl-β-D-thioglucopyranosid (OTG), n-Octyl-β-D-glucopyranosid (OG) Dodecyl-β-D-maltosid (DDM). ( c ) PPY-extracts were tested for their recombination capacity by assembling three DNA fragments with overlapping ends, to generate a recombinant plasmid constitutively expressing a blue chromoprotein. To examine the effects detergents had on the transformation, samples were split after the assembly reaction. One part was transformed into NEB 5-alpha unpurified; the other fraction was purified by silica-column chromatography prior to transformation.

    Techniques Used: Flow Cytometry, Derivative Assay, Recombinant, Plasmid Preparation, Expressing, Transformation Assay, Purification, Column Chromatography

    Comparison of the influence of detergent, autoinduction, post-assembly purification and competency of used bacteria on DNA assembling efficiency. ( a ) In four of the five PPY lysis conditions induction of Redα had moderate effects. Five different detergents were tested on PPY-cells grown with either lactose (Redα un-induced) or arabinose (Redα induced). All assemblies were column-purified before transformation into NEB 5-alpha. Bars indicate standard error of three independent replicates of an assembly reaction in all following graphs. PPY recombinogenic capacity was assessed in three-fragment assemblies. ( b ) Column-purification of the three-fragment-assembly reactions led to markedly increased number of recombinant colonies for all tested detergents. In the case of CHAPS and SB-12, unpurified samples resulted in no colonies. The OTG derived PPY-extract resulted in the highest number of recombinant colonies without purification. All assemblies were arabinose-induced. ( c ) Chemical competency has profound influence on recombination efficiency. OTG prepared PPY-lysate was used in a three-fragment-assembly and transformed into commercial NEB 5-alpha competent E. coli (1 × 10 9 cfu/µg pUC DNA) or the same strain prepared by the Inoue-method 21 (2.3 × 10 6 cfu/µg DNA). ( d ) For convenient readout of the potency of the PPY-extracts PCR-fragments used for the three- and four-way assembly reactions consisted of a blue chromoprotein coding ORF, a kanamycin resistance gene an origin-of-replication (on one fragment for the three-fragment assembly) and a bacterial basal-promoter-fragment. Only successful recombinants could produce blue colonies on kanamycin plates. The PCR-fragments to be assembled had overlapping bases that summed up to about 15 bp overlapping ends.
    Figure Legend Snippet: Comparison of the influence of detergent, autoinduction, post-assembly purification and competency of used bacteria on DNA assembling efficiency. ( a ) In four of the five PPY lysis conditions induction of Redα had moderate effects. Five different detergents were tested on PPY-cells grown with either lactose (Redα un-induced) or arabinose (Redα induced). All assemblies were column-purified before transformation into NEB 5-alpha. Bars indicate standard error of three independent replicates of an assembly reaction in all following graphs. PPY recombinogenic capacity was assessed in three-fragment assemblies. ( b ) Column-purification of the three-fragment-assembly reactions led to markedly increased number of recombinant colonies for all tested detergents. In the case of CHAPS and SB-12, unpurified samples resulted in no colonies. The OTG derived PPY-extract resulted in the highest number of recombinant colonies without purification. All assemblies were arabinose-induced. ( c ) Chemical competency has profound influence on recombination efficiency. OTG prepared PPY-lysate was used in a three-fragment-assembly and transformed into commercial NEB 5-alpha competent E. coli (1 × 10 9 cfu/µg pUC DNA) or the same strain prepared by the Inoue-method 21 (2.3 × 10 6 cfu/µg DNA). ( d ) For convenient readout of the potency of the PPY-extracts PCR-fragments used for the three- and four-way assembly reactions consisted of a blue chromoprotein coding ORF, a kanamycin resistance gene an origin-of-replication (on one fragment for the three-fragment assembly) and a bacterial basal-promoter-fragment. Only successful recombinants could produce blue colonies on kanamycin plates. The PCR-fragments to be assembled had overlapping bases that summed up to about 15 bp overlapping ends.

    Techniques Used: Purification, Lysis, Transformation Assay, Recombinant, Derivative Assay, Polymerase Chain Reaction

    The recombinogenic capacity of OTG extracts from autoinduced PPY and NEB 5-alpha extracts are equivalent or better than PPY-extracts generated by the original protocol. ( a ) Plasmid map of pT7- Hin dIII- ccdB used to assess three-fragment ZeBRα assemblies. Two Hin dIII and two Bsa I sites flank the toxic-placeholder- ccdB , allowing linearization and removal of ccdB . Unique sites are available on either side of ccdB . Chloramphenicol-acetyl-transferase coding gene ( CmR ), is part of the placeholder cassette and prevents ccdB -loss during plasmid propagation. The hatched region encompasses the fragment removed during cloning. ( b ) Map of the vector pT7-GFP antisense resulting from the three-fragment test-assembly of the pT7- Hin dIII- ccdB as recipient for a GFP-ORF and a bacterial promoter containing PCR-fragment, to evaluate the efficacy of the ZeBRα-procedure. Criss-cross lines mark the fusion-sites of the assembled fragments. ( c ) Comparison of the recombination capacity of extracts prepared with OTG or CelLyticB TM from manually induced and autoinduced (denoted as “auto” in the column) PPY-cells and NEB 5-alpha. The iVEC/“transformation-cloning” of the respective fragments is shown as last column. ( d ) Green fluorescent NEB 5-alpha colonies harboring the constitutively GFP-expressing vector pT7-GFP antisense.
    Figure Legend Snippet: The recombinogenic capacity of OTG extracts from autoinduced PPY and NEB 5-alpha extracts are equivalent or better than PPY-extracts generated by the original protocol. ( a ) Plasmid map of pT7- Hin dIII- ccdB used to assess three-fragment ZeBRα assemblies. Two Hin dIII and two Bsa I sites flank the toxic-placeholder- ccdB , allowing linearization and removal of ccdB . Unique sites are available on either side of ccdB . Chloramphenicol-acetyl-transferase coding gene ( CmR ), is part of the placeholder cassette and prevents ccdB -loss during plasmid propagation. The hatched region encompasses the fragment removed during cloning. ( b ) Map of the vector pT7-GFP antisense resulting from the three-fragment test-assembly of the pT7- Hin dIII- ccdB as recipient for a GFP-ORF and a bacterial promoter containing PCR-fragment, to evaluate the efficacy of the ZeBRα-procedure. Criss-cross lines mark the fusion-sites of the assembled fragments. ( c ) Comparison of the recombination capacity of extracts prepared with OTG or CelLyticB TM from manually induced and autoinduced (denoted as “auto” in the column) PPY-cells and NEB 5-alpha. The iVEC/“transformation-cloning” of the respective fragments is shown as last column. ( d ) Green fluorescent NEB 5-alpha colonies harboring the constitutively GFP-expressing vector pT7-GFP antisense.

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

    Rendering the CcdB in the placeholder non-toxic increases the number of GFP – background colonies markedly, showing that ccdB is an essential element if working with non-gel purified vector. ( a ) The pT7- Hin dIII-dead- ccdB differs by a four base-pair deletion in the ccdB -coding sequence from it’s predecessor pT7- Hin dIII- ccdB . The dotted lines encompass the region removed during cloning. ( b ) Sequence alignment of the region encompassing the small deletion in the ccdB -ORF, in pT7- Hin dIII-dead- ccdB compared to the region in pT7- Hin dIII- ccdB and the resulting frameshift rendering the ΔCcdB non-toxic for E. coli NEB 5-alpha. The numbers denote bases in the vector. ( c ) The three-fragment assembly as shown in Fig. 6c uses the pT7-HindIII-dead- ccdB vector and OTG-derived PPY-extract to assemble pT7-GFP antisense analogous to the assemblies shown in Fig. 5 . The percentage of GFP + colonies drops from nearly 100% for pT7- Hin dIII- ccdB to 57% for pT7- Hin dIII-dead- ccdB . ( d ) Image of the mixture of GFP + and GFP − resulting from the assembly (100 µl outgrowth medium spread). The red arrow points at a cluster of GFP − colonies representing un-digested vector that would have to be screened for in a non-model assembly.
    Figure Legend Snippet: Rendering the CcdB in the placeholder non-toxic increases the number of GFP – background colonies markedly, showing that ccdB is an essential element if working with non-gel purified vector. ( a ) The pT7- Hin dIII-dead- ccdB differs by a four base-pair deletion in the ccdB -coding sequence from it’s predecessor pT7- Hin dIII- ccdB . The dotted lines encompass the region removed during cloning. ( b ) Sequence alignment of the region encompassing the small deletion in the ccdB -ORF, in pT7- Hin dIII-dead- ccdB compared to the region in pT7- Hin dIII- ccdB and the resulting frameshift rendering the ΔCcdB non-toxic for E. coli NEB 5-alpha. The numbers denote bases in the vector. ( c ) The three-fragment assembly as shown in Fig. 6c uses the pT7-HindIII-dead- ccdB vector and OTG-derived PPY-extract to assemble pT7-GFP antisense analogous to the assemblies shown in Fig. 5 . The percentage of GFP + colonies drops from nearly 100% for pT7- Hin dIII- ccdB to 57% for pT7- Hin dIII-dead- ccdB . ( d ) Image of the mixture of GFP + and GFP − resulting from the assembly (100 µl outgrowth medium spread). The red arrow points at a cluster of GFP − colonies representing un-digested vector that would have to be screened for in a non-model assembly.

    Techniques Used: Purification, Plasmid Preparation, Sequencing, Clone Assay, Derivative Assay

    10) Product Images from "ZeBRα a universal, multi-fragment DNA-assembly-system with minimal hands-on time requirement"

    Article Title: ZeBRα a universal, multi-fragment DNA-assembly-system with minimal hands-on time requirement

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-39768-0

    Three or four fragments can efficiently be assembled with PPY-cell extracts, while iVEC/“transformation-cloning” with three fragments is markedly less efficient. ( a ) The assembly of four fragments in a single reaction reduces the number of successful assemblies by a factor of two to ten as compared to the three-fragment-assembly. Cell-extracts were prepared from autoinduced PPY cells and the assemblies were purified prior transformation. ( b ) Assembly of three fragments by iVEC/“transformation-cloning” with NEB 5-alpha resulted in roughly 250 recombinant colonies/µg transformed DNA. A significant number of colonies harboring plasmids with defective inserts (grey bar) and PCR-template carry-over (dotted bar) were present on the plates.
    Figure Legend Snippet: Three or four fragments can efficiently be assembled with PPY-cell extracts, while iVEC/“transformation-cloning” with three fragments is markedly less efficient. ( a ) The assembly of four fragments in a single reaction reduces the number of successful assemblies by a factor of two to ten as compared to the three-fragment-assembly. Cell-extracts were prepared from autoinduced PPY cells and the assemblies were purified prior transformation. ( b ) Assembly of three fragments by iVEC/“transformation-cloning” with NEB 5-alpha resulted in roughly 250 recombinant colonies/µg transformed DNA. A significant number of colonies harboring plasmids with defective inserts (grey bar) and PCR-template carry-over (dotted bar) were present on the plates.

    Techniques Used: Clone Assay, Purification, Transformation Assay, Recombinant, Polymerase Chain Reaction

    Strategy for SLiCE optimization and evaluation. ( a ) Flow chart of the optimization process for generating a recombinogenic E. coli lysate. The PPY strain is a DH10B-derivative used to prepare the recombinogenic cell lysate and expresses the coding sequences for Redαβγ. The extracts, derived from arabinose autoinduced PPY-cells, were compared to extracts made from non-induced PPY-cells. ( b ) Structure of the examined non-ionic detergents used to prepare the recombinogenic PPY-extracts, CHAPS, Sulfo-Betain (SB-12), n-Octyl-β-D-thioglucopyranosid (OTG), n-Octyl-β-D-glucopyranosid (OG) Dodecyl-β-D-maltosid (DDM). ( c ) PPY-extracts were tested for their recombination capacity by assembling three DNA fragments with overlapping ends, to generate a recombinant plasmid constitutively expressing a blue chromoprotein. To examine the effects detergents had on the transformation, samples were split after the assembly reaction. One part was transformed into NEB 5-alpha unpurified; the other fraction was purified by silica-column chromatography prior to transformation.
    Figure Legend Snippet: Strategy for SLiCE optimization and evaluation. ( a ) Flow chart of the optimization process for generating a recombinogenic E. coli lysate. The PPY strain is a DH10B-derivative used to prepare the recombinogenic cell lysate and expresses the coding sequences for Redαβγ. The extracts, derived from arabinose autoinduced PPY-cells, were compared to extracts made from non-induced PPY-cells. ( b ) Structure of the examined non-ionic detergents used to prepare the recombinogenic PPY-extracts, CHAPS, Sulfo-Betain (SB-12), n-Octyl-β-D-thioglucopyranosid (OTG), n-Octyl-β-D-glucopyranosid (OG) Dodecyl-β-D-maltosid (DDM). ( c ) PPY-extracts were tested for their recombination capacity by assembling three DNA fragments with overlapping ends, to generate a recombinant plasmid constitutively expressing a blue chromoprotein. To examine the effects detergents had on the transformation, samples were split after the assembly reaction. One part was transformed into NEB 5-alpha unpurified; the other fraction was purified by silica-column chromatography prior to transformation.

    Techniques Used: Flow Cytometry, Derivative Assay, Recombinant, Plasmid Preparation, Expressing, Transformation Assay, Purification, Column Chromatography

    Comparison of the influence of detergent, autoinduction, post-assembly purification and competency of used bacteria on DNA assembling efficiency. ( a ) In four of the five PPY lysis conditions induction of Redα had moderate effects. Five different detergents were tested on PPY-cells grown with either lactose (Redα un-induced) or arabinose (Redα induced). All assemblies were column-purified before transformation into NEB 5-alpha. Bars indicate standard error of three independent replicates of an assembly reaction in all following graphs. PPY recombinogenic capacity was assessed in three-fragment assemblies. ( b ) Column-purification of the three-fragment-assembly reactions led to markedly increased number of recombinant colonies for all tested detergents. In the case of CHAPS and SB-12, unpurified samples resulted in no colonies. The OTG derived PPY-extract resulted in the highest number of recombinant colonies without purification. All assemblies were arabinose-induced. ( c ) Chemical competency has profound influence on recombination efficiency. OTG prepared PPY-lysate was used in a three-fragment-assembly and transformed into commercial NEB 5-alpha competent E. coli (1 × 10 9 cfu/µg pUC DNA) or the same strain prepared by the Inoue-method 21 (2.3 × 10 6 cfu/µg DNA). ( d ) For convenient readout of the potency of the PPY-extracts PCR-fragments used for the three- and four-way assembly reactions consisted of a blue chromoprotein coding ORF, a kanamycin resistance gene an origin-of-replication (on one fragment for the three-fragment assembly) and a bacterial basal-promoter-fragment. Only successful recombinants could produce blue colonies on kanamycin plates. The PCR-fragments to be assembled had overlapping bases that summed up to about 15 bp overlapping ends.
    Figure Legend Snippet: Comparison of the influence of detergent, autoinduction, post-assembly purification and competency of used bacteria on DNA assembling efficiency. ( a ) In four of the five PPY lysis conditions induction of Redα had moderate effects. Five different detergents were tested on PPY-cells grown with either lactose (Redα un-induced) or arabinose (Redα induced). All assemblies were column-purified before transformation into NEB 5-alpha. Bars indicate standard error of three independent replicates of an assembly reaction in all following graphs. PPY recombinogenic capacity was assessed in three-fragment assemblies. ( b ) Column-purification of the three-fragment-assembly reactions led to markedly increased number of recombinant colonies for all tested detergents. In the case of CHAPS and SB-12, unpurified samples resulted in no colonies. The OTG derived PPY-extract resulted in the highest number of recombinant colonies without purification. All assemblies were arabinose-induced. ( c ) Chemical competency has profound influence on recombination efficiency. OTG prepared PPY-lysate was used in a three-fragment-assembly and transformed into commercial NEB 5-alpha competent E. coli (1 × 10 9 cfu/µg pUC DNA) or the same strain prepared by the Inoue-method 21 (2.3 × 10 6 cfu/µg DNA). ( d ) For convenient readout of the potency of the PPY-extracts PCR-fragments used for the three- and four-way assembly reactions consisted of a blue chromoprotein coding ORF, a kanamycin resistance gene an origin-of-replication (on one fragment for the three-fragment assembly) and a bacterial basal-promoter-fragment. Only successful recombinants could produce blue colonies on kanamycin plates. The PCR-fragments to be assembled had overlapping bases that summed up to about 15 bp overlapping ends.

    Techniques Used: Purification, Lysis, Transformation Assay, Recombinant, Derivative Assay, Polymerase Chain Reaction

    The recombinogenic capacity of OTG extracts from autoinduced PPY and NEB 5-alpha extracts are equivalent or better than PPY-extracts generated by the original protocol. ( a ) Plasmid map of pT7- Hin dIII- ccdB used to assess three-fragment ZeBRα assemblies. Two Hin dIII and two Bsa I sites flank the toxic-placeholder- ccdB , allowing linearization and removal of ccdB . Unique sites are available on either side of ccdB . Chloramphenicol-acetyl-transferase coding gene ( CmR ), is part of the placeholder cassette and prevents ccdB -loss during plasmid propagation. The hatched region encompasses the fragment removed during cloning. ( b ) Map of the vector pT7-GFP antisense resulting from the three-fragment test-assembly of the pT7- Hin dIII- ccdB as recipient for a GFP-ORF and a bacterial promoter containing PCR-fragment, to evaluate the efficacy of the ZeBRα-procedure. Criss-cross lines mark the fusion-sites of the assembled fragments. ( c ) Comparison of the recombination capacity of extracts prepared with OTG or CelLyticB TM from manually induced and autoinduced (denoted as “auto” in the column) PPY-cells and NEB 5-alpha. The iVEC/“transformation-cloning” of the respective fragments is shown as last column. ( d ) Green fluorescent NEB 5-alpha colonies harboring the constitutively GFP-expressing vector pT7-GFP antisense.
    Figure Legend Snippet: The recombinogenic capacity of OTG extracts from autoinduced PPY and NEB 5-alpha extracts are equivalent or better than PPY-extracts generated by the original protocol. ( a ) Plasmid map of pT7- Hin dIII- ccdB used to assess three-fragment ZeBRα assemblies. Two Hin dIII and two Bsa I sites flank the toxic-placeholder- ccdB , allowing linearization and removal of ccdB . Unique sites are available on either side of ccdB . Chloramphenicol-acetyl-transferase coding gene ( CmR ), is part of the placeholder cassette and prevents ccdB -loss during plasmid propagation. The hatched region encompasses the fragment removed during cloning. ( b ) Map of the vector pT7-GFP antisense resulting from the three-fragment test-assembly of the pT7- Hin dIII- ccdB as recipient for a GFP-ORF and a bacterial promoter containing PCR-fragment, to evaluate the efficacy of the ZeBRα-procedure. Criss-cross lines mark the fusion-sites of the assembled fragments. ( c ) Comparison of the recombination capacity of extracts prepared with OTG or CelLyticB TM from manually induced and autoinduced (denoted as “auto” in the column) PPY-cells and NEB 5-alpha. The iVEC/“transformation-cloning” of the respective fragments is shown as last column. ( d ) Green fluorescent NEB 5-alpha colonies harboring the constitutively GFP-expressing vector pT7-GFP antisense.

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

    Rendering the CcdB in the placeholder non-toxic increases the number of GFP – background colonies markedly, showing that ccdB is an essential element if working with non-gel purified vector. ( a ) The pT7- Hin dIII-dead- ccdB differs by a four base-pair deletion in the ccdB -coding sequence from it’s predecessor pT7- Hin dIII- ccdB . The dotted lines encompass the region removed during cloning. ( b ) Sequence alignment of the region encompassing the small deletion in the ccdB -ORF, in pT7- Hin dIII-dead- ccdB compared to the region in pT7- Hin dIII- ccdB and the resulting frameshift rendering the ΔCcdB non-toxic for E. coli NEB 5-alpha. The numbers denote bases in the vector. ( c ) The three-fragment assembly as shown in Fig. 6c uses the pT7-HindIII-dead- ccdB vector and OTG-derived PPY-extract to assemble pT7-GFP antisense analogous to the assemblies shown in Fig. 5 . The percentage of GFP + colonies drops from nearly 100% for pT7- Hin dIII- ccdB to 57% for pT7- Hin dIII-dead- ccdB . ( d ) Image of the mixture of GFP + and GFP − resulting from the assembly (100 µl outgrowth medium spread). The red arrow points at a cluster of GFP − colonies representing un-digested vector that would have to be screened for in a non-model assembly.
    Figure Legend Snippet: Rendering the CcdB in the placeholder non-toxic increases the number of GFP – background colonies markedly, showing that ccdB is an essential element if working with non-gel purified vector. ( a ) The pT7- Hin dIII-dead- ccdB differs by a four base-pair deletion in the ccdB -coding sequence from it’s predecessor pT7- Hin dIII- ccdB . The dotted lines encompass the region removed during cloning. ( b ) Sequence alignment of the region encompassing the small deletion in the ccdB -ORF, in pT7- Hin dIII-dead- ccdB compared to the region in pT7- Hin dIII- ccdB and the resulting frameshift rendering the ΔCcdB non-toxic for E. coli NEB 5-alpha. The numbers denote bases in the vector. ( c ) The three-fragment assembly as shown in Fig. 6c uses the pT7-HindIII-dead- ccdB vector and OTG-derived PPY-extract to assemble pT7-GFP antisense analogous to the assemblies shown in Fig. 5 . The percentage of GFP + colonies drops from nearly 100% for pT7- Hin dIII- ccdB to 57% for pT7- Hin dIII-dead- ccdB . ( d ) Image of the mixture of GFP + and GFP − resulting from the assembly (100 µl outgrowth medium spread). The red arrow points at a cluster of GFP − colonies representing un-digested vector that would have to be screened for in a non-model assembly.

    Techniques Used: Purification, Plasmid Preparation, Sequencing, Clone Assay, Derivative Assay

    Related Articles

    Polymerase Chain Reaction:

    Article Title: ZeBRα a universal, multi-fragment DNA-assembly-system with minimal hands-on time requirement
    Article Snippet: .. As in the previous experiments, we assembled three PCR-fragments and transformed half of the column-purified reaction into commercially available NEB 5-alpha (109 cfu/µg) and NEB 5-alpha, made competent by the Inoue-method (2.3 × 106 cfu/µg). .. The competence of the commercial NEB 5-alpha cells was about three hundred times greater than the competence of the cells made competent by the Inoue-method and translated into about 35-times more recombinant colonies (Fig. ).

    Article Title: In Vivo Selection of a Missense Mutation in adeR and Conversion of the Novel blaOXA-164 Gene into blaOXA-58 in Carbapenem-Resistant Acinetobacter baumannii Isolates from a Hospitalized Patient ▿
    Article Snippet: .. PCR products were ligated into EcoRI/BamHI double digested pWH1266 which is an Escherichia coli - Acinetobacter shuttle plasmid ( ) using the In-Fusion dry-down PCR cloning kit (Clontech, Saint-Germain-en-Laye, France) and transformed in E. coli NEB 5-alpha (New England BioLabs, Frankfurt, Germany). .. Plasmids were isolated from transformants and used to transform electrocompetent A. baumannii ATCC 19606 and A. baumannii ATCC 17978 selected on Mueller-Hinton agar containing 100 μg/ml ticarcillin.

    Clone Assay:

    Article Title: FabV/Triclosan Is an Antibiotic-Free and Cost-Effective Selection System for Efficient Maintenance of High and Medium -Copy Number Plasmids in Escherichia coli
    Article Snippet: .. Bacterial strains and culture conditions The E . coli strains DH5α (NEB #C2987) and JM109 (NEB #E4107) were used for cloning whereas BL21(DE3) (NEB # C2527) along with the other two E . coli strains was used for subsequent experiments. ..

    Article Title: In Vivo Selection of a Missense Mutation in adeR and Conversion of the Novel blaOXA-164 Gene into blaOXA-58 in Carbapenem-Resistant Acinetobacter baumannii Isolates from a Hospitalized Patient ▿
    Article Snippet: .. PCR products were ligated into EcoRI/BamHI double digested pWH1266 which is an Escherichia coli - Acinetobacter shuttle plasmid ( ) using the In-Fusion dry-down PCR cloning kit (Clontech, Saint-Germain-en-Laye, France) and transformed in E. coli NEB 5-alpha (New England BioLabs, Frankfurt, Germany). .. Plasmids were isolated from transformants and used to transform electrocompetent A. baumannii ATCC 19606 and A. baumannii ATCC 17978 selected on Mueller-Hinton agar containing 100 μg/ml ticarcillin.

    Transformation Assay:

    Article Title: ZeBRα a universal, multi-fragment DNA-assembly-system with minimal hands-on time requirement
    Article Snippet: .. As in the previous experiments, we assembled three PCR-fragments and transformed half of the column-purified reaction into commercially available NEB 5-alpha (109 cfu/µg) and NEB 5-alpha, made competent by the Inoue-method (2.3 × 106 cfu/µg). .. The competence of the commercial NEB 5-alpha cells was about three hundred times greater than the competence of the cells made competent by the Inoue-method and translated into about 35-times more recombinant colonies (Fig. ).

    Article Title: In Vivo Selection of a Missense Mutation in adeR and Conversion of the Novel blaOXA-164 Gene into blaOXA-58 in Carbapenem-Resistant Acinetobacter baumannii Isolates from a Hospitalized Patient ▿
    Article Snippet: .. PCR products were ligated into EcoRI/BamHI double digested pWH1266 which is an Escherichia coli - Acinetobacter shuttle plasmid ( ) using the In-Fusion dry-down PCR cloning kit (Clontech, Saint-Germain-en-Laye, France) and transformed in E. coli NEB 5-alpha (New England BioLabs, Frankfurt, Germany). .. Plasmids were isolated from transformants and used to transform electrocompetent A. baumannii ATCC 19606 and A. baumannii ATCC 17978 selected on Mueller-Hinton agar containing 100 μg/ml ticarcillin.

    Article Title: ZeBRα a universal, multi-fragment DNA-assembly-system with minimal hands-on time requirement
    Article Snippet: .. Transformation of NEB 5-alpha with assembled Plasmids and measuring the recombination capacity of the PPY extracts Frozen chemically competent NEB 5-alpha (DH5α–derivative, NEB) cells (2.3 × 106 cfu/µg) were thawed on ice. .. A volume corresponding to 200 ng total DNA from the purified assembly was added to 100 μl bacterial suspension and incubated on ice for 30 minutes.

    Derivative Assay:

    Article Title: ZeBRα a universal, multi-fragment DNA-assembly-system with minimal hands-on time requirement
    Article Snippet: .. To our surprise the SLiCE-extract with OTG and other tested detergents derived from NEB 5-alpha worked equivalently well as the respective extract from the recombinase-expressing PPY-strain. .. The high efficiency of the NEB 5-alpha derived SLiCE was unexpected since we have previously shown (Fig. ) that PPY derived extracts from recombinase-induced cells outperformed their un-induced PPY-counterparts.

    Article Title: ZeBRα a universal, multi-fragment DNA-assembly-system with minimal hands-on time requirement
    Article Snippet: .. To our surprise the OTG-extract derived from NEB 5-alpha resulted in the highest number of recombinants 62389 cfu/µg (N = 3) (Fig. ). .. Finally, the iVEC/“transformation-cloning”-method resulted in 1061 cfu/µg (N = 3) transformants, but as previously observed for this method (Fig. ) the percentage of GFP expressing colonies was with 30% of the total number of colonies on the plate markedly lower than for the plasmids assembled by bacterial extracts in vitro , reaching 99–100% GFP-expressing colonies.

    Plasmid Preparation:

    Article Title: In Vivo Selection of a Missense Mutation in adeR and Conversion of the Novel blaOXA-164 Gene into blaOXA-58 in Carbapenem-Resistant Acinetobacter baumannii Isolates from a Hospitalized Patient ▿
    Article Snippet: .. PCR products were ligated into EcoRI/BamHI double digested pWH1266 which is an Escherichia coli - Acinetobacter shuttle plasmid ( ) using the In-Fusion dry-down PCR cloning kit (Clontech, Saint-Germain-en-Laye, France) and transformed in E. coli NEB 5-alpha (New England BioLabs, Frankfurt, Germany). .. Plasmids were isolated from transformants and used to transform electrocompetent A. baumannii ATCC 19606 and A. baumannii ATCC 17978 selected on Mueller-Hinton agar containing 100 μg/ml ticarcillin.

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    New England Biolabs e coli neb 5 alpha
    Imipenem and meropenem Etests for E. coli NEB <t>5-alpha</t> carrying pWH1266:: bla OXA-164 and showing imipenem and meropenem heteroresistance.
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    Imipenem and meropenem Etests for E. coli NEB 5-alpha carrying pWH1266:: bla OXA-164 and showing imipenem and meropenem heteroresistance.

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: In Vivo Selection of a Missense Mutation in adeR and Conversion of the Novel blaOXA-164 Gene into blaOXA-58 in Carbapenem-Resistant Acinetobacter baumannii Isolates from a Hospitalized Patient ▿

    doi: 10.1128/AAC.00598-10

    Figure Lengend Snippet: Imipenem and meropenem Etests for E. coli NEB 5-alpha carrying pWH1266:: bla OXA-164 and showing imipenem and meropenem heteroresistance.

    Article Snippet: PCR products were ligated into EcoRI/BamHI double digested pWH1266 which is an Escherichia coli - Acinetobacter shuttle plasmid ( ) using the In-Fusion dry-down PCR cloning kit (Clontech, Saint-Germain-en-Laye, France) and transformed in E. coli NEB 5-alpha (New England BioLabs, Frankfurt, Germany).

    Techniques:

    Three or four fragments can efficiently be assembled with PPY-cell extracts, while iVEC/“transformation-cloning” with three fragments is markedly less efficient. ( a ) The assembly of four fragments in a single reaction reduces the number of successful assemblies by a factor of two to ten as compared to the three-fragment-assembly. Cell-extracts were prepared from autoinduced PPY cells and the assemblies were purified prior transformation. ( b ) Assembly of three fragments by iVEC/“transformation-cloning” with NEB 5-alpha resulted in roughly 250 recombinant colonies/µg transformed DNA. A significant number of colonies harboring plasmids with defective inserts (grey bar) and PCR-template carry-over (dotted bar) were present on the plates.

    Journal: Scientific Reports

    Article Title: ZeBRα a universal, multi-fragment DNA-assembly-system with minimal hands-on time requirement

    doi: 10.1038/s41598-019-39768-0

    Figure Lengend Snippet: Three or four fragments can efficiently be assembled with PPY-cell extracts, while iVEC/“transformation-cloning” with three fragments is markedly less efficient. ( a ) The assembly of four fragments in a single reaction reduces the number of successful assemblies by a factor of two to ten as compared to the three-fragment-assembly. Cell-extracts were prepared from autoinduced PPY cells and the assemblies were purified prior transformation. ( b ) Assembly of three fragments by iVEC/“transformation-cloning” with NEB 5-alpha resulted in roughly 250 recombinant colonies/µg transformed DNA. A significant number of colonies harboring plasmids with defective inserts (grey bar) and PCR-template carry-over (dotted bar) were present on the plates.

    Article Snippet: To our surprise the OTG-extract derived from NEB 5-alpha resulted in the highest number of recombinants 62389 cfu/µg (N = 3) (Fig. ).

    Techniques: Clone Assay, Purification, Transformation Assay, Recombinant, Polymerase Chain Reaction

    Strategy for SLiCE optimization and evaluation. ( a ) Flow chart of the optimization process for generating a recombinogenic E. coli lysate. The PPY strain is a DH10B-derivative used to prepare the recombinogenic cell lysate and expresses the coding sequences for Redαβγ. The extracts, derived from arabinose autoinduced PPY-cells, were compared to extracts made from non-induced PPY-cells. ( b ) Structure of the examined non-ionic detergents used to prepare the recombinogenic PPY-extracts, CHAPS, Sulfo-Betain (SB-12), n-Octyl-β-D-thioglucopyranosid (OTG), n-Octyl-β-D-glucopyranosid (OG) Dodecyl-β-D-maltosid (DDM). ( c ) PPY-extracts were tested for their recombination capacity by assembling three DNA fragments with overlapping ends, to generate a recombinant plasmid constitutively expressing a blue chromoprotein. To examine the effects detergents had on the transformation, samples were split after the assembly reaction. One part was transformed into NEB 5-alpha unpurified; the other fraction was purified by silica-column chromatography prior to transformation.

    Journal: Scientific Reports

    Article Title: ZeBRα a universal, multi-fragment DNA-assembly-system with minimal hands-on time requirement

    doi: 10.1038/s41598-019-39768-0

    Figure Lengend Snippet: Strategy for SLiCE optimization and evaluation. ( a ) Flow chart of the optimization process for generating a recombinogenic E. coli lysate. The PPY strain is a DH10B-derivative used to prepare the recombinogenic cell lysate and expresses the coding sequences for Redαβγ. The extracts, derived from arabinose autoinduced PPY-cells, were compared to extracts made from non-induced PPY-cells. ( b ) Structure of the examined non-ionic detergents used to prepare the recombinogenic PPY-extracts, CHAPS, Sulfo-Betain (SB-12), n-Octyl-β-D-thioglucopyranosid (OTG), n-Octyl-β-D-glucopyranosid (OG) Dodecyl-β-D-maltosid (DDM). ( c ) PPY-extracts were tested for their recombination capacity by assembling three DNA fragments with overlapping ends, to generate a recombinant plasmid constitutively expressing a blue chromoprotein. To examine the effects detergents had on the transformation, samples were split after the assembly reaction. One part was transformed into NEB 5-alpha unpurified; the other fraction was purified by silica-column chromatography prior to transformation.

    Article Snippet: To our surprise the OTG-extract derived from NEB 5-alpha resulted in the highest number of recombinants 62389 cfu/µg (N = 3) (Fig. ).

    Techniques: Flow Cytometry, Derivative Assay, Recombinant, Plasmid Preparation, Expressing, Transformation Assay, Purification, Column Chromatography

    Comparison of the influence of detergent, autoinduction, post-assembly purification and competency of used bacteria on DNA assembling efficiency. ( a ) In four of the five PPY lysis conditions induction of Redα had moderate effects. Five different detergents were tested on PPY-cells grown with either lactose (Redα un-induced) or arabinose (Redα induced). All assemblies were column-purified before transformation into NEB 5-alpha. Bars indicate standard error of three independent replicates of an assembly reaction in all following graphs. PPY recombinogenic capacity was assessed in three-fragment assemblies. ( b ) Column-purification of the three-fragment-assembly reactions led to markedly increased number of recombinant colonies for all tested detergents. In the case of CHAPS and SB-12, unpurified samples resulted in no colonies. The OTG derived PPY-extract resulted in the highest number of recombinant colonies without purification. All assemblies were arabinose-induced. ( c ) Chemical competency has profound influence on recombination efficiency. OTG prepared PPY-lysate was used in a three-fragment-assembly and transformed into commercial NEB 5-alpha competent E. coli (1 × 10 9 cfu/µg pUC DNA) or the same strain prepared by the Inoue-method 21 (2.3 × 10 6 cfu/µg DNA). ( d ) For convenient readout of the potency of the PPY-extracts PCR-fragments used for the three- and four-way assembly reactions consisted of a blue chromoprotein coding ORF, a kanamycin resistance gene an origin-of-replication (on one fragment for the three-fragment assembly) and a bacterial basal-promoter-fragment. Only successful recombinants could produce blue colonies on kanamycin plates. The PCR-fragments to be assembled had overlapping bases that summed up to about 15 bp overlapping ends.

    Journal: Scientific Reports

    Article Title: ZeBRα a universal, multi-fragment DNA-assembly-system with minimal hands-on time requirement

    doi: 10.1038/s41598-019-39768-0

    Figure Lengend Snippet: Comparison of the influence of detergent, autoinduction, post-assembly purification and competency of used bacteria on DNA assembling efficiency. ( a ) In four of the five PPY lysis conditions induction of Redα had moderate effects. Five different detergents were tested on PPY-cells grown with either lactose (Redα un-induced) or arabinose (Redα induced). All assemblies were column-purified before transformation into NEB 5-alpha. Bars indicate standard error of three independent replicates of an assembly reaction in all following graphs. PPY recombinogenic capacity was assessed in three-fragment assemblies. ( b ) Column-purification of the three-fragment-assembly reactions led to markedly increased number of recombinant colonies for all tested detergents. In the case of CHAPS and SB-12, unpurified samples resulted in no colonies. The OTG derived PPY-extract resulted in the highest number of recombinant colonies without purification. All assemblies were arabinose-induced. ( c ) Chemical competency has profound influence on recombination efficiency. OTG prepared PPY-lysate was used in a three-fragment-assembly and transformed into commercial NEB 5-alpha competent E. coli (1 × 10 9 cfu/µg pUC DNA) or the same strain prepared by the Inoue-method 21 (2.3 × 10 6 cfu/µg DNA). ( d ) For convenient readout of the potency of the PPY-extracts PCR-fragments used for the three- and four-way assembly reactions consisted of a blue chromoprotein coding ORF, a kanamycin resistance gene an origin-of-replication (on one fragment for the three-fragment assembly) and a bacterial basal-promoter-fragment. Only successful recombinants could produce blue colonies on kanamycin plates. The PCR-fragments to be assembled had overlapping bases that summed up to about 15 bp overlapping ends.

    Article Snippet: To our surprise the OTG-extract derived from NEB 5-alpha resulted in the highest number of recombinants 62389 cfu/µg (N = 3) (Fig. ).

    Techniques: Purification, Lysis, Transformation Assay, Recombinant, Derivative Assay, Polymerase Chain Reaction

    The recombinogenic capacity of OTG extracts from autoinduced PPY and NEB 5-alpha extracts are equivalent or better than PPY-extracts generated by the original protocol. ( a ) Plasmid map of pT7- Hin dIII- ccdB used to assess three-fragment ZeBRα assemblies. Two Hin dIII and two Bsa I sites flank the toxic-placeholder- ccdB , allowing linearization and removal of ccdB . Unique sites are available on either side of ccdB . Chloramphenicol-acetyl-transferase coding gene ( CmR ), is part of the placeholder cassette and prevents ccdB -loss during plasmid propagation. The hatched region encompasses the fragment removed during cloning. ( b ) Map of the vector pT7-GFP antisense resulting from the three-fragment test-assembly of the pT7- Hin dIII- ccdB as recipient for a GFP-ORF and a bacterial promoter containing PCR-fragment, to evaluate the efficacy of the ZeBRα-procedure. Criss-cross lines mark the fusion-sites of the assembled fragments. ( c ) Comparison of the recombination capacity of extracts prepared with OTG or CelLyticB TM from manually induced and autoinduced (denoted as “auto” in the column) PPY-cells and NEB 5-alpha. The iVEC/“transformation-cloning” of the respective fragments is shown as last column. ( d ) Green fluorescent NEB 5-alpha colonies harboring the constitutively GFP-expressing vector pT7-GFP antisense.

    Journal: Scientific Reports

    Article Title: ZeBRα a universal, multi-fragment DNA-assembly-system with minimal hands-on time requirement

    doi: 10.1038/s41598-019-39768-0

    Figure Lengend Snippet: The recombinogenic capacity of OTG extracts from autoinduced PPY and NEB 5-alpha extracts are equivalent or better than PPY-extracts generated by the original protocol. ( a ) Plasmid map of pT7- Hin dIII- ccdB used to assess three-fragment ZeBRα assemblies. Two Hin dIII and two Bsa I sites flank the toxic-placeholder- ccdB , allowing linearization and removal of ccdB . Unique sites are available on either side of ccdB . Chloramphenicol-acetyl-transferase coding gene ( CmR ), is part of the placeholder cassette and prevents ccdB -loss during plasmid propagation. The hatched region encompasses the fragment removed during cloning. ( b ) Map of the vector pT7-GFP antisense resulting from the three-fragment test-assembly of the pT7- Hin dIII- ccdB as recipient for a GFP-ORF and a bacterial promoter containing PCR-fragment, to evaluate the efficacy of the ZeBRα-procedure. Criss-cross lines mark the fusion-sites of the assembled fragments. ( c ) Comparison of the recombination capacity of extracts prepared with OTG or CelLyticB TM from manually induced and autoinduced (denoted as “auto” in the column) PPY-cells and NEB 5-alpha. The iVEC/“transformation-cloning” of the respective fragments is shown as last column. ( d ) Green fluorescent NEB 5-alpha colonies harboring the constitutively GFP-expressing vector pT7-GFP antisense.

    Article Snippet: To our surprise the OTG-extract derived from NEB 5-alpha resulted in the highest number of recombinants 62389 cfu/µg (N = 3) (Fig. ).

    Techniques: Generated, Plasmid Preparation, Chloramphenicol Acetyltransferase Assay, Clone Assay, Polymerase Chain Reaction, Expressing

    Rendering the CcdB in the placeholder non-toxic increases the number of GFP – background colonies markedly, showing that ccdB is an essential element if working with non-gel purified vector. ( a ) The pT7- Hin dIII-dead- ccdB differs by a four base-pair deletion in the ccdB -coding sequence from it’s predecessor pT7- Hin dIII- ccdB . The dotted lines encompass the region removed during cloning. ( b ) Sequence alignment of the region encompassing the small deletion in the ccdB -ORF, in pT7- Hin dIII-dead- ccdB compared to the region in pT7- Hin dIII- ccdB and the resulting frameshift rendering the ΔCcdB non-toxic for E. coli NEB 5-alpha. The numbers denote bases in the vector. ( c ) The three-fragment assembly as shown in Fig. 6c uses the pT7-HindIII-dead- ccdB vector and OTG-derived PPY-extract to assemble pT7-GFP antisense analogous to the assemblies shown in Fig. 5 . The percentage of GFP + colonies drops from nearly 100% for pT7- Hin dIII- ccdB to 57% for pT7- Hin dIII-dead- ccdB . ( d ) Image of the mixture of GFP + and GFP − resulting from the assembly (100 µl outgrowth medium spread). The red arrow points at a cluster of GFP − colonies representing un-digested vector that would have to be screened for in a non-model assembly.

    Journal: Scientific Reports

    Article Title: ZeBRα a universal, multi-fragment DNA-assembly-system with minimal hands-on time requirement

    doi: 10.1038/s41598-019-39768-0

    Figure Lengend Snippet: Rendering the CcdB in the placeholder non-toxic increases the number of GFP – background colonies markedly, showing that ccdB is an essential element if working with non-gel purified vector. ( a ) The pT7- Hin dIII-dead- ccdB differs by a four base-pair deletion in the ccdB -coding sequence from it’s predecessor pT7- Hin dIII- ccdB . The dotted lines encompass the region removed during cloning. ( b ) Sequence alignment of the region encompassing the small deletion in the ccdB -ORF, in pT7- Hin dIII-dead- ccdB compared to the region in pT7- Hin dIII- ccdB and the resulting frameshift rendering the ΔCcdB non-toxic for E. coli NEB 5-alpha. The numbers denote bases in the vector. ( c ) The three-fragment assembly as shown in Fig. 6c uses the pT7-HindIII-dead- ccdB vector and OTG-derived PPY-extract to assemble pT7-GFP antisense analogous to the assemblies shown in Fig. 5 . The percentage of GFP + colonies drops from nearly 100% for pT7- Hin dIII- ccdB to 57% for pT7- Hin dIII-dead- ccdB . ( d ) Image of the mixture of GFP + and GFP − resulting from the assembly (100 µl outgrowth medium spread). The red arrow points at a cluster of GFP − colonies representing un-digested vector that would have to be screened for in a non-model assembly.

    Article Snippet: To our surprise the OTG-extract derived from NEB 5-alpha resulted in the highest number of recombinants 62389 cfu/µg (N = 3) (Fig. ).

    Techniques: Purification, Plasmid Preparation, Sequencing, Clone Assay, Derivative Assay