purexpress kit  (New England Biolabs)


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    Structured Review

    New England Biolabs purexpress kit
    mRNA stability and translation of the hfq mRNA. (A) The WT and Δ csrA strains were grown to log phase in TMH-gal, and RNA was isolated from samples taken at 0 min (prerifampin) and after the addition of 400 μg/ml rifampin. Relative hfq mRNA levels remaining at the indicated time points were quantified using RT-qPCR. The amount of hfq mRNA in each strain at 0 min relative to rifampin addition was set to 100%. The percent mRNA remaining thereafter was plotted versus time as semilogarithmic graphs. The mean ± SEM percent mRNA from four independent experiments is shown. (B) In vitro translational assays were performed with the <t>PURExpress</t> kit using translational fusions transcripts of hfq-gfp and hmsT-gfp (negative control) expressed from a T7 promoter. The mean ± SD fold change in HmsT-GFP and Hfq-GFP signal between samples in the presence or absence of CsrA was derived from three technical replicates of the immunodot blot. One representative dot blot is shown.
    Purexpress Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/purexpress kit/product/New England Biolabs
    Average 97 stars, based on 28 article reviews
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    Images

    1) Product Images from "CsrA Enhances Cyclic-di-GMP Biosynthesis and Yersinia pestis Biofilm Blockage of the Flea Foregut by Alleviating Hfq-Dependent Repression of the hmsT mRNA"

    Article Title: CsrA Enhances Cyclic-di-GMP Biosynthesis and Yersinia pestis Biofilm Blockage of the Flea Foregut by Alleviating Hfq-Dependent Repression of the hmsT mRNA

    Journal: mBio

    doi: 10.1128/mBio.01358-21

    mRNA stability and translation of the hfq mRNA. (A) The WT and Δ csrA strains were grown to log phase in TMH-gal, and RNA was isolated from samples taken at 0 min (prerifampin) and after the addition of 400 μg/ml rifampin. Relative hfq mRNA levels remaining at the indicated time points were quantified using RT-qPCR. The amount of hfq mRNA in each strain at 0 min relative to rifampin addition was set to 100%. The percent mRNA remaining thereafter was plotted versus time as semilogarithmic graphs. The mean ± SEM percent mRNA from four independent experiments is shown. (B) In vitro translational assays were performed with the PURExpress kit using translational fusions transcripts of hfq-gfp and hmsT-gfp (negative control) expressed from a T7 promoter. The mean ± SD fold change in HmsT-GFP and Hfq-GFP signal between samples in the presence or absence of CsrA was derived from three technical replicates of the immunodot blot. One representative dot blot is shown.
    Figure Legend Snippet: mRNA stability and translation of the hfq mRNA. (A) The WT and Δ csrA strains were grown to log phase in TMH-gal, and RNA was isolated from samples taken at 0 min (prerifampin) and after the addition of 400 μg/ml rifampin. Relative hfq mRNA levels remaining at the indicated time points were quantified using RT-qPCR. The amount of hfq mRNA in each strain at 0 min relative to rifampin addition was set to 100%. The percent mRNA remaining thereafter was plotted versus time as semilogarithmic graphs. The mean ± SEM percent mRNA from four independent experiments is shown. (B) In vitro translational assays were performed with the PURExpress kit using translational fusions transcripts of hfq-gfp and hmsT-gfp (negative control) expressed from a T7 promoter. The mean ± SD fold change in HmsT-GFP and Hfq-GFP signal between samples in the presence or absence of CsrA was derived from three technical replicates of the immunodot blot. One representative dot blot is shown.

    Techniques Used: Isolation, Quantitative RT-PCR, In Vitro, Negative Control, Derivative Assay, Dot Blot

    2) Product Images from "CsrA-Mediated Translational Activation of ymdA Expression in Escherichia coli"

    Article Title: CsrA-Mediated Translational Activation of ymdA Expression in Escherichia coli

    Journal: mBio

    doi: 10.1128/mBio.00849-20

    CsrA activates ymdA translation by facilitating 30S ribosomal subunit binding. (A) CsrA and 30S ribosome toeprint analysis of ymdA leader RNA. The presence of CsrA, tRNA fMet , 30S ribosomal subunit (30S Rib), and/or reverse transcriptase (SSIII RT) is indicated above each lane. The positions of CsrA (CsrA), 30S ribosomal subunit (30S Rib), and SD-sequestering hairpin (SD hairpin) toeprints are indicated with arrows. Sequencing lanes A, C, G, and U are marked, and lane numbers are shown at the bottom of the gel. Numbering is with respect to the start of ymdA translation. (B) Coupled transcription-translation reactions were performed using the PURExpress kit with wild-type (WT), BS1 mutant (BS1) and BS2 mutant (BS2) DNA templates containing ymdA'-'lacZ translational fusions expressed from a T7 promoter. A negative control that was shown previously to be unaffected by CsrA was also used ( 41 ). Purified CsrA was added prior to the start of each reaction. β-Galactosidase activity was normalized to 0 μM CsrA for each template. Values are averages ± standard deviations (error bars) from three experiments.
    Figure Legend Snippet: CsrA activates ymdA translation by facilitating 30S ribosomal subunit binding. (A) CsrA and 30S ribosome toeprint analysis of ymdA leader RNA. The presence of CsrA, tRNA fMet , 30S ribosomal subunit (30S Rib), and/or reverse transcriptase (SSIII RT) is indicated above each lane. The positions of CsrA (CsrA), 30S ribosomal subunit (30S Rib), and SD-sequestering hairpin (SD hairpin) toeprints are indicated with arrows. Sequencing lanes A, C, G, and U are marked, and lane numbers are shown at the bottom of the gel. Numbering is with respect to the start of ymdA translation. (B) Coupled transcription-translation reactions were performed using the PURExpress kit with wild-type (WT), BS1 mutant (BS1) and BS2 mutant (BS2) DNA templates containing ymdA'-'lacZ translational fusions expressed from a T7 promoter. A negative control that was shown previously to be unaffected by CsrA was also used ( 41 ). Purified CsrA was added prior to the start of each reaction. β-Galactosidase activity was normalized to 0 μM CsrA for each template. Values are averages ± standard deviations (error bars) from three experiments.

    Techniques Used: Binding Assay, Sequencing, Mutagenesis, Negative Control, Purification, Activity Assay

    3) Product Images from "Accurate target identification for Mycobacterium tuberculosis endoribonuclease toxins requires expression in their native host"

    Article Title: Accurate target identification for Mycobacterium tuberculosis endoribonuclease toxins requires expression in their native host

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-41548-9

    VapC-mt11 inhibits translation in vitro and in M. smegmatis . ( A ) The PURExpress translation reaction was incubated with (+) or without (−) VapC-mt11. Production of the control DHFR template was assayed (yellow arrow). Uncropped image shown in Supplementary Information Fig. 1 ( B ) [ 35 S]-Methionine incorporation in M. smegmatis mc 2 155 cells grown to exponential phase and split into uninduced (−VapC-mt11) and induced (+VapC-mt11) cultures. Cell aliquots were collected for up to 6 h post induction. Equivalent amounts of cell lysate (resuspended in appropriate Laemmli buffer volumes to normalize for differences in OD 600 ) were subjected to SDS-PAGE and visualized on a phosphorimager. Uncropped image shown in Supplementary Information Fig. 1 .
    Figure Legend Snippet: VapC-mt11 inhibits translation in vitro and in M. smegmatis . ( A ) The PURExpress translation reaction was incubated with (+) or without (−) VapC-mt11. Production of the control DHFR template was assayed (yellow arrow). Uncropped image shown in Supplementary Information Fig. 1 ( B ) [ 35 S]-Methionine incorporation in M. smegmatis mc 2 155 cells grown to exponential phase and split into uninduced (−VapC-mt11) and induced (+VapC-mt11) cultures. Cell aliquots were collected for up to 6 h post induction. Equivalent amounts of cell lysate (resuspended in appropriate Laemmli buffer volumes to normalize for differences in OD 600 ) were subjected to SDS-PAGE and visualized on a phosphorimager. Uncropped image shown in Supplementary Information Fig. 1 .

    Techniques Used: In Vitro, Incubation, SDS Page

    4) Product Images from "Escherichia coli ItaT is a type II toxin that inhibits translation by acetylating isoleucyl-tRNAIle"

    Article Title: Escherichia coli ItaT is a type II toxin that inhibits translation by acetylating isoleucyl-tRNAIle

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky560

    ItaT inhibits translation by acetylation of tRNA. ( A ) Activity of firefly luciferase synthesized in in vitro coupled transcription-translation PURExpress system in the absence or presence of ItaT, acetyl-CoA (AcCoA) or both. The graph shows the relative luminescence of samples obtained from three independent experiments expressed in arbitrary units, AU. The error bars represent standard deviation. ( B ) SDS-PAGE analysis of DHFR produced in the transcription-translation reaction described in (A). Reactions were supplemented with [ 14 C] acetyl-CoA and carried out in the absence (left) or presence of ItaT (right). Reaction products were visualized with Coomassie blue staining. The position of the DHFR protein band is indicated by the red arrowhead on the left. Lane M shows the molecular weight markers. ( C ) Analysis of tRNAs extracted from the samples shown in (B) by acid-urea PAGE followed by methylene blue staining (top panel) and autoradiography (lower panel). The position of tRNAs on the gel is indicated by the black arrowhead on the right.
    Figure Legend Snippet: ItaT inhibits translation by acetylation of tRNA. ( A ) Activity of firefly luciferase synthesized in in vitro coupled transcription-translation PURExpress system in the absence or presence of ItaT, acetyl-CoA (AcCoA) or both. The graph shows the relative luminescence of samples obtained from three independent experiments expressed in arbitrary units, AU. The error bars represent standard deviation. ( B ) SDS-PAGE analysis of DHFR produced in the transcription-translation reaction described in (A). Reactions were supplemented with [ 14 C] acetyl-CoA and carried out in the absence (left) or presence of ItaT (right). Reaction products were visualized with Coomassie blue staining. The position of the DHFR protein band is indicated by the red arrowhead on the left. Lane M shows the molecular weight markers. ( C ) Analysis of tRNAs extracted from the samples shown in (B) by acid-urea PAGE followed by methylene blue staining (top panel) and autoradiography (lower panel). The position of tRNAs on the gel is indicated by the black arrowhead on the right.

    Techniques Used: Activity Assay, Luciferase, Synthesized, In Vitro, Standard Deviation, SDS Page, Produced, Staining, Molecular Weight, Polyacrylamide Gel Electrophoresis, Autoradiography

    5) Product Images from "Escherichia coli ItaT is a type II toxin that inhibits translation by acetylating isoleucyl-tRNAIle"

    Article Title: Escherichia coli ItaT is a type II toxin that inhibits translation by acetylating isoleucyl-tRNAIle

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky560

    ItaT inhibits translation by acetylation of tRNA. ( A ) Activity of firefly luciferase synthesized in in vitro coupled transcription-translation PURExpress system in the absence or presence of ItaT, acetyl-CoA (AcCoA) or both. The graph shows the relative luminescence of samples obtained from three independent experiments expressed in arbitrary units, AU. The error bars represent standard deviation. ( B ) SDS-PAGE analysis of DHFR produced in the transcription-translation reaction described in (A). Reactions were supplemented with [ 14 C] acetyl-CoA and carried out in the absence (left) or presence of ItaT (right). Reaction products were visualized with Coomassie blue staining. The position of the DHFR protein band is indicated by the red arrowhead on the left. Lane M shows the molecular weight markers. ( C ) Analysis of tRNAs extracted from the samples shown in (B) by acid-urea PAGE followed by methylene blue staining (top panel) and autoradiography (lower panel). The position of tRNAs on the gel is indicated by the black arrowhead on the right.
    Figure Legend Snippet: ItaT inhibits translation by acetylation of tRNA. ( A ) Activity of firefly luciferase synthesized in in vitro coupled transcription-translation PURExpress system in the absence or presence of ItaT, acetyl-CoA (AcCoA) or both. The graph shows the relative luminescence of samples obtained from three independent experiments expressed in arbitrary units, AU. The error bars represent standard deviation. ( B ) SDS-PAGE analysis of DHFR produced in the transcription-translation reaction described in (A). Reactions were supplemented with [ 14 C] acetyl-CoA and carried out in the absence (left) or presence of ItaT (right). Reaction products were visualized with Coomassie blue staining. The position of the DHFR protein band is indicated by the red arrowhead on the left. Lane M shows the molecular weight markers. ( C ) Analysis of tRNAs extracted from the samples shown in (B) by acid-urea PAGE followed by methylene blue staining (top panel) and autoradiography (lower panel). The position of tRNAs on the gel is indicated by the black arrowhead on the right.

    Techniques Used: Activity Assay, Luciferase, Synthesized, In Vitro, Standard Deviation, SDS Page, Produced, Staining, Molecular Weight, Polyacrylamide Gel Electrophoresis, Autoradiography

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  • 97
    New England Biolabs vitro protein synthesis kit
    ShdA homologues exhibit nuclease activity in vitro. (a) Cells carrying plasmid pBAD18 encoding ShdA II-His6 were grown for 5 hrs in the presence of 0.2 % L-arabinose. Cells were fractionated to produce soluble and membrane samples and analysed by immunoblot with antibodies to the His6 tag, GroEL (cytoplasmic control) and TatA (membrane control). (b-e) In vitro DNAse activity assays using (b) ShdA II 138-524 (c) ShdA I 140-526 (d) ShdA III 135-523 and (e) ShdA IV 135-521 . ShdA proteins and DHFR were synthesised using the <t>cell-free</t> <t>PURExpress</t> <t>kit</t> (NEB). DNAse activity was tested against 10 ng of input DNA. DNA types tested were phage DNA, E. coli MG1655 chromosomal DNA and plasmid (pSG483) DNA. For ShdA I 140-526 , ShdA III 135-523 and ShdA II 138-524 phage DNA was from ϕSipho. For ShdA IV 135-521 phage DNA was from ϕAlma.
    Vitro Protein Synthesis Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 79 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/vitro protein synthesis kit/product/New England Biolabs
    Average 97 stars, based on 79 article reviews
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    ShdA homologues exhibit nuclease activity in vitro. (a) Cells carrying plasmid pBAD18 encoding ShdA II-His6 were grown for 5 hrs in the presence of 0.2 % L-arabinose. Cells were fractionated to produce soluble and membrane samples and analysed by immunoblot with antibodies to the His6 tag, GroEL (cytoplasmic control) and TatA (membrane control). (b-e) In vitro DNAse activity assays using (b) ShdA II 138-524 (c) ShdA I 140-526 (d) ShdA III 135-523 and (e) ShdA IV 135-521 . ShdA proteins and DHFR were synthesised using the cell-free PURExpress kit (NEB). DNAse activity was tested against 10 ng of input DNA. DNA types tested were phage DNA, E. coli MG1655 chromosomal DNA and plasmid (pSG483) DNA. For ShdA I 140-526 , ShdA III 135-523 and ShdA II 138-524 phage DNA was from ϕSipho. For ShdA IV 135-521 phage DNA was from ϕAlma.

    Journal: bioRxiv

    Article Title: Shield co-opts an RmuC domain to mediate phage defence across Pseudomonas species

    doi: 10.1101/2022.11.04.515146

    Figure Lengend Snippet: ShdA homologues exhibit nuclease activity in vitro. (a) Cells carrying plasmid pBAD18 encoding ShdA II-His6 were grown for 5 hrs in the presence of 0.2 % L-arabinose. Cells were fractionated to produce soluble and membrane samples and analysed by immunoblot with antibodies to the His6 tag, GroEL (cytoplasmic control) and TatA (membrane control). (b-e) In vitro DNAse activity assays using (b) ShdA II 138-524 (c) ShdA I 140-526 (d) ShdA III 135-523 and (e) ShdA IV 135-521 . ShdA proteins and DHFR were synthesised using the cell-free PURExpress kit (NEB). DNAse activity was tested against 10 ng of input DNA. DNA types tested were phage DNA, E. coli MG1655 chromosomal DNA and plasmid (pSG483) DNA. For ShdA I 140-526 , ShdA III 135-523 and ShdA II 138-524 phage DNA was from ϕSipho. For ShdA IV 135-521 phage DNA was from ϕAlma.

    Article Snippet: Cell free protein synthesis and Nuclease assay The RmuC domains of ShdA I, ShdA II, ShdA III and ShdA IV homologues were synthesised in vitro using the PURExpress cell-free transcription/translation kit (NEB), from PCR products.

    Techniques: Activity Assay, In Vitro, Plasmid Preparation