pbad33  (New England Biolabs)


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    Structured Review

    New England Biolabs pbad33
    Molecular cloning of chiA-3 and expression of ChiA-3-6xHis. (a): Schematic of cloning strategy used to amplify and insert chiA-3 directionally into the <t>pBAD33</t> multiple cloning site (MCS), under the arabinose-inducible P BAD promoter, and to incorporate a C-terminal 6xHis tag as a translational fusion. A linker sequence was not incorporated between the C-terminus of ChiA-3 and the 6xHis tag. Figures are not to scale. (b): InstantBlue-stained acrylamide gel of proteins present in supernatants and cell pellet lysates from cultures grown at 23 °C supplemented with arabinose (induction +) or glucose (induction -). No induced bands were easily discerned. (c): Western immunoblot produced from an identically-loaded acrylamide gel to that presented in (b), run in parallel with the gel in (b), and probed with an α-6xHis antibody (see Methods). A band corresponding to the expected molecular weight of ChiA-3-6xHis (48.51 kDa) was detected in the cell pellet lysate of E. coli harbouring pMJD157 only (plasmid +). This size is consistent with the retention of the fusion protein without the cleavage of the putative signal sequence. Protein ladders: NEB #P7719S and #P7717S. EV = empty vector (pBAD33).
    Pbad33, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pbad33/product/New England Biolabs
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pbad33 - by Bioz Stars, 2022-07
    86/100 stars

    Images

    1) Product Images from "gbpA and chiA genes are not uniformly distributed amongst diverse Vibrio cholerae"

    Article Title: gbpA and chiA genes are not uniformly distributed amongst diverse Vibrio cholerae

    Journal: bioRxiv

    doi: 10.1101/2021.02.11.430729

    Molecular cloning of chiA-3 and expression of ChiA-3-6xHis. (a): Schematic of cloning strategy used to amplify and insert chiA-3 directionally into the pBAD33 multiple cloning site (MCS), under the arabinose-inducible P BAD promoter, and to incorporate a C-terminal 6xHis tag as a translational fusion. A linker sequence was not incorporated between the C-terminus of ChiA-3 and the 6xHis tag. Figures are not to scale. (b): InstantBlue-stained acrylamide gel of proteins present in supernatants and cell pellet lysates from cultures grown at 23 °C supplemented with arabinose (induction +) or glucose (induction -). No induced bands were easily discerned. (c): Western immunoblot produced from an identically-loaded acrylamide gel to that presented in (b), run in parallel with the gel in (b), and probed with an α-6xHis antibody (see Methods). A band corresponding to the expected molecular weight of ChiA-3-6xHis (48.51 kDa) was detected in the cell pellet lysate of E. coli harbouring pMJD157 only (plasmid +). This size is consistent with the retention of the fusion protein without the cleavage of the putative signal sequence. Protein ladders: NEB #P7719S and #P7717S. EV = empty vector (pBAD33).
    Figure Legend Snippet: Molecular cloning of chiA-3 and expression of ChiA-3-6xHis. (a): Schematic of cloning strategy used to amplify and insert chiA-3 directionally into the pBAD33 multiple cloning site (MCS), under the arabinose-inducible P BAD promoter, and to incorporate a C-terminal 6xHis tag as a translational fusion. A linker sequence was not incorporated between the C-terminus of ChiA-3 and the 6xHis tag. Figures are not to scale. (b): InstantBlue-stained acrylamide gel of proteins present in supernatants and cell pellet lysates from cultures grown at 23 °C supplemented with arabinose (induction +) or glucose (induction -). No induced bands were easily discerned. (c): Western immunoblot produced from an identically-loaded acrylamide gel to that presented in (b), run in parallel with the gel in (b), and probed with an α-6xHis antibody (see Methods). A band corresponding to the expected molecular weight of ChiA-3-6xHis (48.51 kDa) was detected in the cell pellet lysate of E. coli harbouring pMJD157 only (plasmid +). This size is consistent with the retention of the fusion protein without the cleavage of the putative signal sequence. Protein ladders: NEB #P7719S and #P7717S. EV = empty vector (pBAD33).

    Techniques Used: Molecular Cloning, Expressing, Clone Assay, Sequencing, Staining, Acrylamide Gel Assay, Western Blot, Produced, Molecular Weight, Plasmid Preparation

    2) Product Images from "gbpA and chiA genes are not uniformly distributed amongst diverse Vibrio cholerae"

    Article Title: gbpA and chiA genes are not uniformly distributed amongst diverse Vibrio cholerae

    Journal: bioRxiv

    doi: 10.1101/2021.02.11.430729

    Molecular cloning of chiA-3 and expression of ChiA-3-6xHis. (a): Schematic of cloning strategy used to amplify and insert chiA-3 directionally into the pBAD33 multiple cloning site (MCS), under the arabinose-inducible P BAD promoter, and to incorporate a C-terminal 6xHis tag as a translational fusion. A linker sequence was not incorporated between the C-terminus of ChiA-3 and the 6xHis tag. Figures are not to scale. (b): InstantBlue-stained acrylamide gel of proteins present in supernatants and cell pellet lysates from cultures grown at 23 °C supplemented with arabinose (induction +) or glucose (induction -). No induced bands were easily discerned. (c): Western immunoblot produced from an identically-loaded acrylamide gel to that presented in (b), run in parallel with the gel in (b), and probed with an α-6xHis antibody (see Methods). A band corresponding to the expected molecular weight of ChiA-3-6xHis (48.51 kDa) was detected in the cell pellet lysate of E. coli harbouring pMJD157 only (plasmid +). This size is consistent with the retention of the fusion protein without the cleavage of the putative signal sequence. Protein ladders: NEB #P7719S and #P7717S. EV = empty vector (pBAD33).
    Figure Legend Snippet: Molecular cloning of chiA-3 and expression of ChiA-3-6xHis. (a): Schematic of cloning strategy used to amplify and insert chiA-3 directionally into the pBAD33 multiple cloning site (MCS), under the arabinose-inducible P BAD promoter, and to incorporate a C-terminal 6xHis tag as a translational fusion. A linker sequence was not incorporated between the C-terminus of ChiA-3 and the 6xHis tag. Figures are not to scale. (b): InstantBlue-stained acrylamide gel of proteins present in supernatants and cell pellet lysates from cultures grown at 23 °C supplemented with arabinose (induction +) or glucose (induction -). No induced bands were easily discerned. (c): Western immunoblot produced from an identically-loaded acrylamide gel to that presented in (b), run in parallel with the gel in (b), and probed with an α-6xHis antibody (see Methods). A band corresponding to the expected molecular weight of ChiA-3-6xHis (48.51 kDa) was detected in the cell pellet lysate of E. coli harbouring pMJD157 only (plasmid +). This size is consistent with the retention of the fusion protein without the cleavage of the putative signal sequence. Protein ladders: NEB #P7719S and #P7717S. EV = empty vector (pBAD33).

    Techniques Used: Molecular Cloning, Expressing, Clone Assay, Sequencing, Staining, Acrylamide Gel Assay, Western Blot, Produced, Molecular Weight, Plasmid Preparation

    3) Product Images from "Characterization of a toxin-antitoxin system in Mycobacterium tuberculosis suggests neutralization by phosphorylation as the antitoxicity mechanism"

    Article Title: Characterization of a toxin-antitoxin system in Mycobacterium tuberculosis suggests neutralization by phosphorylation as the antitoxicity mechanism

    Journal: Communications Biology

    doi: 10.1038/s42003-020-0941-1

    Rv1044-Rv1045 of H37Rv constitutes a bona fide TA system. a Diagram of the genetic organization of the Rv1044-Rv1045 operon in the H37Rv genome (not to scale). The location of the genes is indicated. b Toxicity and antitoxicity assay of Rv1044-Rv1045 pair in E. coli . The expression of the pBAD33-c-6His-Rv1045 plasmid encoding TglT-His resulted in cell growth arrest; the toxicity was neutralized when the pET28-n-6His-Rv1044 plasmid encoding His-TakA was co-expressed. See plasmid details in Supplementary Table 2 . c Growth curves of BL21 cells containing plasmids encoding the toxin (pBAD33-c-6His-Rv1045) and the antitoxin (pET28-n-6His-Rv1044), or the toxin with the empty vector. For all experiments, the bacteria were induced when OD 600 reached 0.2, which was set as hour 0. The OD 600 was then measured at the indicated time points. Red curve, the toxin was induced (+Ara) first and the antitoxin was induced (+IPTG) 2 h later; green curve, the toxin was induced (+Ara) but the antitoxin was not induced (−IPTG); blue curve, BL21 cells containing pBAD33-c-6His-Rv1045 and the empty pET28a but without induction (−Ara); black curve, BL21 cells containing pBAD33-c-6His-Rv1045 and the empty pET28a with induction (+Ara). Data shown are mean OD 600 value ± SD ( n = 3). d The interaction between TglT and TakA was detected by a Co-IP experiment. e – g NTP binding assays demonstrate that toxin TglT preferentially binds GTP. In the upper two native-PAGEs, TglT forms a complex with radioactively labeled GTP, but not with ATP, CTP or UTP. The concentration of all NTPs in the assays was constant (3.3 nM). The concentration of TglT in the GTP binding assay started from the highest 16 μM (right side) to the lowest 2 nM (left side) by two-fold serial dilutions; the concentrations of TglT used in other NTP binding assays were 16 μM, 8 μM, 4 μM and 2 μM. The bottom native-PAGE shows competition binding. The concentrations of TglT and [α- 32 P] labeled GTP were 8 μM and 3.3 nM, whereas the concentrations of the cold competitors were 0.5 μM, 4 μM and 16 μM, respectively. Source data are provided as a Supplementary Data 2.
    Figure Legend Snippet: Rv1044-Rv1045 of H37Rv constitutes a bona fide TA system. a Diagram of the genetic organization of the Rv1044-Rv1045 operon in the H37Rv genome (not to scale). The location of the genes is indicated. b Toxicity and antitoxicity assay of Rv1044-Rv1045 pair in E. coli . The expression of the pBAD33-c-6His-Rv1045 plasmid encoding TglT-His resulted in cell growth arrest; the toxicity was neutralized when the pET28-n-6His-Rv1044 plasmid encoding His-TakA was co-expressed. See plasmid details in Supplementary Table 2 . c Growth curves of BL21 cells containing plasmids encoding the toxin (pBAD33-c-6His-Rv1045) and the antitoxin (pET28-n-6His-Rv1044), or the toxin with the empty vector. For all experiments, the bacteria were induced when OD 600 reached 0.2, which was set as hour 0. The OD 600 was then measured at the indicated time points. Red curve, the toxin was induced (+Ara) first and the antitoxin was induced (+IPTG) 2 h later; green curve, the toxin was induced (+Ara) but the antitoxin was not induced (−IPTG); blue curve, BL21 cells containing pBAD33-c-6His-Rv1045 and the empty pET28a but without induction (−Ara); black curve, BL21 cells containing pBAD33-c-6His-Rv1045 and the empty pET28a with induction (+Ara). Data shown are mean OD 600 value ± SD ( n = 3). d The interaction between TglT and TakA was detected by a Co-IP experiment. e – g NTP binding assays demonstrate that toxin TglT preferentially binds GTP. In the upper two native-PAGEs, TglT forms a complex with radioactively labeled GTP, but not with ATP, CTP or UTP. The concentration of all NTPs in the assays was constant (3.3 nM). The concentration of TglT in the GTP binding assay started from the highest 16 μM (right side) to the lowest 2 nM (left side) by two-fold serial dilutions; the concentrations of TglT used in other NTP binding assays were 16 μM, 8 μM, 4 μM and 2 μM. The bottom native-PAGE shows competition binding. The concentrations of TglT and [α- 32 P] labeled GTP were 8 μM and 3.3 nM, whereas the concentrations of the cold competitors were 0.5 μM, 4 μM and 16 μM, respectively. Source data are provided as a Supplementary Data 2.

    Techniques Used: Expressing, Plasmid Preparation, Acetylene Reduction Assay, Co-Immunoprecipitation Assay, Binding Assay, Labeling, Concentration Assay, GTP Binding Assay, Clear Native PAGE

    4) Product Images from "Characterization of a toxin-antitoxin system in Mycobacterium tuberculosis suggests neutralization by phosphorylation as the antitoxicity mechanism"

    Article Title: Characterization of a toxin-antitoxin system in Mycobacterium tuberculosis suggests neutralization by phosphorylation as the antitoxicity mechanism

    Journal: Communications Biology

    doi: 10.1038/s42003-020-0941-1

    Rv1044-Rv1045 of H37Rv constitutes a bona fide TA system. a Diagram of the genetic organization of the Rv1044-Rv1045 operon in the H37Rv genome (not to scale). The location of the genes is indicated. b Toxicity and antitoxicity assay of Rv1044-Rv1045 pair in E. coli . The expression of the pBAD33-c-6His-Rv1045 plasmid encoding TglT-His resulted in cell growth arrest; the toxicity was neutralized when the pET28-n-6His-Rv1044 plasmid encoding His-TakA was co-expressed. See plasmid details in Supplementary Table 2 . c Growth curves of BL21 cells containing plasmids encoding the toxin (pBAD33-c-6His-Rv1045) and the antitoxin (pET28-n-6His-Rv1044), or the toxin with the empty vector. For all experiments, the bacteria were induced when OD 600 reached 0.2, which was set as hour 0. The OD 600 was then measured at the indicated time points. Red curve, the toxin was induced (+Ara) first and the antitoxin was induced (+IPTG) 2 h later; green curve, the toxin was induced (+Ara) but the antitoxin was not induced (−IPTG); blue curve, BL21 cells containing pBAD33-c-6His-Rv1045 and the empty pET28a but without induction (−Ara); black curve, BL21 cells containing pBAD33-c-6His-Rv1045 and the empty pET28a with induction (+Ara). Data shown are mean OD 600 value ± SD ( n = 3). d The interaction between TglT and TakA was detected by a Co-IP experiment. e – g NTP binding assays demonstrate that toxin TglT preferentially binds GTP. In the upper two native-PAGEs, TglT forms a complex with radioactively labeled GTP, but not with ATP, CTP or UTP. The concentration of all NTPs in the assays was constant (3.3 nM). The concentration of TglT in the GTP binding assay started from the highest 16 μM (right side) to the lowest 2 nM (left side) by two-fold serial dilutions; the concentrations of TglT used in other NTP binding assays were 16 μM, 8 μM, 4 μM and 2 μM. The bottom native-PAGE shows competition binding. The concentrations of TglT and [α- 32 P] labeled GTP were 8 μM and 3.3 nM, whereas the concentrations of the cold competitors were 0.5 μM, 4 μM and 16 μM, respectively. Source data are provided as a Supplementary Data 2.
    Figure Legend Snippet: Rv1044-Rv1045 of H37Rv constitutes a bona fide TA system. a Diagram of the genetic organization of the Rv1044-Rv1045 operon in the H37Rv genome (not to scale). The location of the genes is indicated. b Toxicity and antitoxicity assay of Rv1044-Rv1045 pair in E. coli . The expression of the pBAD33-c-6His-Rv1045 plasmid encoding TglT-His resulted in cell growth arrest; the toxicity was neutralized when the pET28-n-6His-Rv1044 plasmid encoding His-TakA was co-expressed. See plasmid details in Supplementary Table 2 . c Growth curves of BL21 cells containing plasmids encoding the toxin (pBAD33-c-6His-Rv1045) and the antitoxin (pET28-n-6His-Rv1044), or the toxin with the empty vector. For all experiments, the bacteria were induced when OD 600 reached 0.2, which was set as hour 0. The OD 600 was then measured at the indicated time points. Red curve, the toxin was induced (+Ara) first and the antitoxin was induced (+IPTG) 2 h later; green curve, the toxin was induced (+Ara) but the antitoxin was not induced (−IPTG); blue curve, BL21 cells containing pBAD33-c-6His-Rv1045 and the empty pET28a but without induction (−Ara); black curve, BL21 cells containing pBAD33-c-6His-Rv1045 and the empty pET28a with induction (+Ara). Data shown are mean OD 600 value ± SD ( n = 3). d The interaction between TglT and TakA was detected by a Co-IP experiment. e – g NTP binding assays demonstrate that toxin TglT preferentially binds GTP. In the upper two native-PAGEs, TglT forms a complex with radioactively labeled GTP, but not with ATP, CTP or UTP. The concentration of all NTPs in the assays was constant (3.3 nM). The concentration of TglT in the GTP binding assay started from the highest 16 μM (right side) to the lowest 2 nM (left side) by two-fold serial dilutions; the concentrations of TglT used in other NTP binding assays were 16 μM, 8 μM, 4 μM and 2 μM. The bottom native-PAGE shows competition binding. The concentrations of TglT and [α- 32 P] labeled GTP were 8 μM and 3.3 nM, whereas the concentrations of the cold competitors were 0.5 μM, 4 μM and 16 μM, respectively. Source data are provided as a Supplementary Data 2.

    Techniques Used: Expressing, Plasmid Preparation, Acetylene Reduction Assay, Co-Immunoprecipitation Assay, Binding Assay, Labeling, Concentration Assay, GTP Binding Assay, Clear Native PAGE

    5) Product Images from "Insights into the Molecular Basis of L-Form Formation and Survival in Escherichia coli"

    Article Title: Insights into the Molecular Basis of L-Form Formation and Survival in Escherichia coli

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0007316

    Complementation of deletion mutants restore L-form colony growth. A, B E. coli BW25113 alone and BW25113 transformed with empty vector pBAD33 showing “fried egg” morphology on LIM without 0.2% arabinose. C, D E. coli BW25113 alone and BW25113 transformed with empty vector pBAD33 showing multiple “fried egg” colonies on LIM with 0.2% arabinose. E, G, I, K ruvA mutant transformed with empty vector pBAD33 showing no growth on LIM with and without 0.2% arabinose. F, J ruvA mutant complemented with wild type gene showing no growth on LIM without 0.2% arabinose. H, L, M, N, O ruvA, rcsB, fur, and smpA mutants complemented with their respective wild type genes showing restoration of “fried egg” colony growth on LIM with 0.2% arabinose. All microscopic images are by Hoffman modulation and scale bars are 100 µm unless specified. Images I through L are gross views of LIM plates.
    Figure Legend Snippet: Complementation of deletion mutants restore L-form colony growth. A, B E. coli BW25113 alone and BW25113 transformed with empty vector pBAD33 showing “fried egg” morphology on LIM without 0.2% arabinose. C, D E. coli BW25113 alone and BW25113 transformed with empty vector pBAD33 showing multiple “fried egg” colonies on LIM with 0.2% arabinose. E, G, I, K ruvA mutant transformed with empty vector pBAD33 showing no growth on LIM with and without 0.2% arabinose. F, J ruvA mutant complemented with wild type gene showing no growth on LIM without 0.2% arabinose. H, L, M, N, O ruvA, rcsB, fur, and smpA mutants complemented with their respective wild type genes showing restoration of “fried egg” colony growth on LIM with 0.2% arabinose. All microscopic images are by Hoffman modulation and scale bars are 100 µm unless specified. Images I through L are gross views of LIM plates.

    Techniques Used: Transformation Assay, Plasmid Preparation, Mutagenesis

    6) Product Images from "Characterization of a toxin-antitoxin system in Mycobacterium tuberculosis suggests neutralization by phosphorylation as the antitoxicity mechanism"

    Article Title: Characterization of a toxin-antitoxin system in Mycobacterium tuberculosis suggests neutralization by phosphorylation as the antitoxicity mechanism

    Journal: Communications Biology

    doi: 10.1038/s42003-020-0941-1

    Rv1044-Rv1045 of H37Rv constitutes a bona fide TA system. a Diagram of the genetic organization of the Rv1044-Rv1045 operon in the H37Rv genome (not to scale). The location of the genes is indicated. b Toxicity and antitoxicity assay of Rv1044-Rv1045 pair in E. coli . The expression of the pBAD33-c-6His-Rv1045 plasmid encoding TglT-His resulted in cell growth arrest; the toxicity was neutralized when the pET28-n-6His-Rv1044 plasmid encoding His-TakA was co-expressed. See plasmid details in Supplementary Table 2 . c Growth curves of BL21 cells containing plasmids encoding the toxin (pBAD33-c-6His-Rv1045) and the antitoxin (pET28-n-6His-Rv1044), or the toxin with the empty vector. For all experiments, the bacteria were induced when OD 600 reached 0.2, which was set as hour 0. The OD 600 was then measured at the indicated time points. Red curve, the toxin was induced (+Ara) first and the antitoxin was induced (+IPTG) 2 h later; green curve, the toxin was induced (+Ara) but the antitoxin was not induced (−IPTG); blue curve, BL21 cells containing pBAD33-c-6His-Rv1045 and the empty pET28a but without induction (−Ara); black curve, BL21 cells containing pBAD33-c-6His-Rv1045 and the empty pET28a with induction (+Ara). Data shown are mean OD 600 value ± SD ( n = 3). d The interaction between TglT and TakA was detected by a Co-IP experiment. e – g NTP binding assays demonstrate that toxin TglT preferentially binds GTP. In the upper two native-PAGEs, TglT forms a complex with radioactively labeled GTP, but not with ATP, CTP or UTP. The concentration of all NTPs in the assays was constant (3.3 nM). The concentration of TglT in the GTP binding assay started from the highest 16 μM (right side) to the lowest 2 nM (left side) by two-fold serial dilutions; the concentrations of TglT used in other NTP binding assays were 16 μM, 8 μM, 4 μM and 2 μM. The bottom native-PAGE shows competition binding. The concentrations of TglT and [α- 32 P] labeled GTP were 8 μM and 3.3 nM, whereas the concentrations of the cold competitors were 0.5 μM, 4 μM and 16 μM, respectively. Source data are provided as a Supplementary Data 2.
    Figure Legend Snippet: Rv1044-Rv1045 of H37Rv constitutes a bona fide TA system. a Diagram of the genetic organization of the Rv1044-Rv1045 operon in the H37Rv genome (not to scale). The location of the genes is indicated. b Toxicity and antitoxicity assay of Rv1044-Rv1045 pair in E. coli . The expression of the pBAD33-c-6His-Rv1045 plasmid encoding TglT-His resulted in cell growth arrest; the toxicity was neutralized when the pET28-n-6His-Rv1044 plasmid encoding His-TakA was co-expressed. See plasmid details in Supplementary Table 2 . c Growth curves of BL21 cells containing plasmids encoding the toxin (pBAD33-c-6His-Rv1045) and the antitoxin (pET28-n-6His-Rv1044), or the toxin with the empty vector. For all experiments, the bacteria were induced when OD 600 reached 0.2, which was set as hour 0. The OD 600 was then measured at the indicated time points. Red curve, the toxin was induced (+Ara) first and the antitoxin was induced (+IPTG) 2 h later; green curve, the toxin was induced (+Ara) but the antitoxin was not induced (−IPTG); blue curve, BL21 cells containing pBAD33-c-6His-Rv1045 and the empty pET28a but without induction (−Ara); black curve, BL21 cells containing pBAD33-c-6His-Rv1045 and the empty pET28a with induction (+Ara). Data shown are mean OD 600 value ± SD ( n = 3). d The interaction between TglT and TakA was detected by a Co-IP experiment. e – g NTP binding assays demonstrate that toxin TglT preferentially binds GTP. In the upper two native-PAGEs, TglT forms a complex with radioactively labeled GTP, but not with ATP, CTP or UTP. The concentration of all NTPs in the assays was constant (3.3 nM). The concentration of TglT in the GTP binding assay started from the highest 16 μM (right side) to the lowest 2 nM (left side) by two-fold serial dilutions; the concentrations of TglT used in other NTP binding assays were 16 μM, 8 μM, 4 μM and 2 μM. The bottom native-PAGE shows competition binding. The concentrations of TglT and [α- 32 P] labeled GTP were 8 μM and 3.3 nM, whereas the concentrations of the cold competitors were 0.5 μM, 4 μM and 16 μM, respectively. Source data are provided as a Supplementary Data 2.

    Techniques Used: Expressing, Plasmid Preparation, Acetylene Reduction Assay, Co-Immunoprecipitation Assay, Binding Assay, Labeling, Concentration Assay, GTP Binding Assay, Clear Native PAGE

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    New England Biolabs pbad33
    Molecular cloning of chiA-3 and expression of ChiA-3-6xHis. (a): Schematic of cloning strategy used to amplify and insert chiA-3 directionally into the <t>pBAD33</t> multiple cloning site (MCS), under the arabinose-inducible P BAD promoter, and to incorporate a C-terminal 6xHis tag as a translational fusion. A linker sequence was not incorporated between the C-terminus of ChiA-3 and the 6xHis tag. Figures are not to scale. (b): InstantBlue-stained acrylamide gel of proteins present in supernatants and cell pellet lysates from cultures grown at 23 °C supplemented with arabinose (induction +) or glucose (induction -). No induced bands were easily discerned. (c): Western immunoblot produced from an identically-loaded acrylamide gel to that presented in (b), run in parallel with the gel in (b), and probed with an α-6xHis antibody (see Methods). A band corresponding to the expected molecular weight of ChiA-3-6xHis (48.51 kDa) was detected in the cell pellet lysate of E. coli harbouring pMJD157 only (plasmid +). This size is consistent with the retention of the fusion protein without the cleavage of the putative signal sequence. Protein ladders: NEB #P7719S and #P7717S. EV = empty vector (pBAD33).
    Pbad33, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pbad33/product/New England Biolabs
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pbad33 - by Bioz Stars, 2022-07
    95/100 stars
      Buy from Supplier

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    Molecular cloning of chiA-3 and expression of ChiA-3-6xHis. (a): Schematic of cloning strategy used to amplify and insert chiA-3 directionally into the pBAD33 multiple cloning site (MCS), under the arabinose-inducible P BAD promoter, and to incorporate a C-terminal 6xHis tag as a translational fusion. A linker sequence was not incorporated between the C-terminus of ChiA-3 and the 6xHis tag. Figures are not to scale. (b): InstantBlue-stained acrylamide gel of proteins present in supernatants and cell pellet lysates from cultures grown at 23 °C supplemented with arabinose (induction +) or glucose (induction -). No induced bands were easily discerned. (c): Western immunoblot produced from an identically-loaded acrylamide gel to that presented in (b), run in parallel with the gel in (b), and probed with an α-6xHis antibody (see Methods). A band corresponding to the expected molecular weight of ChiA-3-6xHis (48.51 kDa) was detected in the cell pellet lysate of E. coli harbouring pMJD157 only (plasmid +). This size is consistent with the retention of the fusion protein without the cleavage of the putative signal sequence. Protein ladders: NEB #P7719S and #P7717S. EV = empty vector (pBAD33).

    Journal: bioRxiv

    Article Title: gbpA and chiA genes are not uniformly distributed amongst diverse Vibrio cholerae

    doi: 10.1101/2021.02.11.430729

    Figure Lengend Snippet: Molecular cloning of chiA-3 and expression of ChiA-3-6xHis. (a): Schematic of cloning strategy used to amplify and insert chiA-3 directionally into the pBAD33 multiple cloning site (MCS), under the arabinose-inducible P BAD promoter, and to incorporate a C-terminal 6xHis tag as a translational fusion. A linker sequence was not incorporated between the C-terminus of ChiA-3 and the 6xHis tag. Figures are not to scale. (b): InstantBlue-stained acrylamide gel of proteins present in supernatants and cell pellet lysates from cultures grown at 23 °C supplemented with arabinose (induction +) or glucose (induction -). No induced bands were easily discerned. (c): Western immunoblot produced from an identically-loaded acrylamide gel to that presented in (b), run in parallel with the gel in (b), and probed with an α-6xHis antibody (see Methods). A band corresponding to the expected molecular weight of ChiA-3-6xHis (48.51 kDa) was detected in the cell pellet lysate of E. coli harbouring pMJD157 only (plasmid +). This size is consistent with the retention of the fusion protein without the cleavage of the putative signal sequence. Protein ladders: NEB #P7719S and #P7717S. EV = empty vector (pBAD33).

    Article Snippet: The amplicon was purified and digested using 30 units of SacI-HF and SalI-HF (NEB, #R3156S and R3138S respectively) at 37 °C for 45 min. pBAD33 was similarly treated with SacI-HF and SalI-HF, and after 15 min incubation at 37 °C, the plasmid digestion was supplemented with 1.5 units of recombinant shrimp alkaline phosphatase (rSAP; NEB #M0371S).

    Techniques: Molecular Cloning, Expressing, Clone Assay, Sequencing, Staining, Acrylamide Gel Assay, Western Blot, Produced, Molecular Weight, Plasmid Preparation

    Rv1044-Rv1045 of H37Rv constitutes a bona fide TA system. a Diagram of the genetic organization of the Rv1044-Rv1045 operon in the H37Rv genome (not to scale). The location of the genes is indicated. b Toxicity and antitoxicity assay of Rv1044-Rv1045 pair in E. coli . The expression of the pBAD33-c-6His-Rv1045 plasmid encoding TglT-His resulted in cell growth arrest; the toxicity was neutralized when the pET28-n-6His-Rv1044 plasmid encoding His-TakA was co-expressed. See plasmid details in Supplementary Table 2 . c Growth curves of BL21 cells containing plasmids encoding the toxin (pBAD33-c-6His-Rv1045) and the antitoxin (pET28-n-6His-Rv1044), or the toxin with the empty vector. For all experiments, the bacteria were induced when OD 600 reached 0.2, which was set as hour 0. The OD 600 was then measured at the indicated time points. Red curve, the toxin was induced (+Ara) first and the antitoxin was induced (+IPTG) 2 h later; green curve, the toxin was induced (+Ara) but the antitoxin was not induced (−IPTG); blue curve, BL21 cells containing pBAD33-c-6His-Rv1045 and the empty pET28a but without induction (−Ara); black curve, BL21 cells containing pBAD33-c-6His-Rv1045 and the empty pET28a with induction (+Ara). Data shown are mean OD 600 value ± SD ( n = 3). d The interaction between TglT and TakA was detected by a Co-IP experiment. e – g NTP binding assays demonstrate that toxin TglT preferentially binds GTP. In the upper two native-PAGEs, TglT forms a complex with radioactively labeled GTP, but not with ATP, CTP or UTP. The concentration of all NTPs in the assays was constant (3.3 nM). The concentration of TglT in the GTP binding assay started from the highest 16 μM (right side) to the lowest 2 nM (left side) by two-fold serial dilutions; the concentrations of TglT used in other NTP binding assays were 16 μM, 8 μM, 4 μM and 2 μM. The bottom native-PAGE shows competition binding. The concentrations of TglT and [α- 32 P] labeled GTP were 8 μM and 3.3 nM, whereas the concentrations of the cold competitors were 0.5 μM, 4 μM and 16 μM, respectively. Source data are provided as a Supplementary Data 2.

    Journal: Communications Biology

    Article Title: Characterization of a toxin-antitoxin system in Mycobacterium tuberculosis suggests neutralization by phosphorylation as the antitoxicity mechanism

    doi: 10.1038/s42003-020-0941-1

    Figure Lengend Snippet: Rv1044-Rv1045 of H37Rv constitutes a bona fide TA system. a Diagram of the genetic organization of the Rv1044-Rv1045 operon in the H37Rv genome (not to scale). The location of the genes is indicated. b Toxicity and antitoxicity assay of Rv1044-Rv1045 pair in E. coli . The expression of the pBAD33-c-6His-Rv1045 plasmid encoding TglT-His resulted in cell growth arrest; the toxicity was neutralized when the pET28-n-6His-Rv1044 plasmid encoding His-TakA was co-expressed. See plasmid details in Supplementary Table 2 . c Growth curves of BL21 cells containing plasmids encoding the toxin (pBAD33-c-6His-Rv1045) and the antitoxin (pET28-n-6His-Rv1044), or the toxin with the empty vector. For all experiments, the bacteria were induced when OD 600 reached 0.2, which was set as hour 0. The OD 600 was then measured at the indicated time points. Red curve, the toxin was induced (+Ara) first and the antitoxin was induced (+IPTG) 2 h later; green curve, the toxin was induced (+Ara) but the antitoxin was not induced (−IPTG); blue curve, BL21 cells containing pBAD33-c-6His-Rv1045 and the empty pET28a but without induction (−Ara); black curve, BL21 cells containing pBAD33-c-6His-Rv1045 and the empty pET28a with induction (+Ara). Data shown are mean OD 600 value ± SD ( n = 3). d The interaction between TglT and TakA was detected by a Co-IP experiment. e – g NTP binding assays demonstrate that toxin TglT preferentially binds GTP. In the upper two native-PAGEs, TglT forms a complex with radioactively labeled GTP, but not with ATP, CTP or UTP. The concentration of all NTPs in the assays was constant (3.3 nM). The concentration of TglT in the GTP binding assay started from the highest 16 μM (right side) to the lowest 2 nM (left side) by two-fold serial dilutions; the concentrations of TglT used in other NTP binding assays were 16 μM, 8 μM, 4 μM and 2 μM. The bottom native-PAGE shows competition binding. The concentrations of TglT and [α- 32 P] labeled GTP were 8 μM and 3.3 nM, whereas the concentrations of the cold competitors were 0.5 μM, 4 μM and 16 μM, respectively. Source data are provided as a Supplementary Data 2.

    Article Snippet: Toxicity and antitoxicity assay Rv1045 and Rv1044 genes were amplified and cloned into pBAD33 and pET28a vectors respectively, encoding TglT-His and His-TakA (Supplementary Table ).

    Techniques: Expressing, Plasmid Preparation, Acetylene Reduction Assay, Co-Immunoprecipitation Assay, Binding Assay, Labeling, Concentration Assay, GTP Binding Assay, Clear Native PAGE