e coli strain dh5α  (Thermo Fisher)


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    Structured Review

    Thermo Fisher e coli strain dh5α
    ΦTE-F expresses ToxI RNA during infection. (A) Upper panel; an S1-nuclease protection assay was used to detect ToxI levels from a ToxIN plasmid during ΦTE wt infection, using an antisense probe against the full 5.5 repeat ToxI sequence [46] . The antisense ToxI-probe was first hybridised to 10 µg of total RNA prepared from Pba ToxIN (pMJ4) at different times after ΦTE infection, and then followed by S1-nuclease treatment. Numbers (+) indicate the time (min) after infection or (−) without the addition of phage. Pba ToxIN-FS (pTA47) and Pba serve as positive and negative controls, respectively. A non-hybridized S1-digested probe (+S1) serves as a further negative control. <t>DH5α</t> 1.5 repeats (pTA96), a non-S1 digested probe (−S1) and an in vitro transcribed Hammerhead ribozyme (HHRz), which cleaves itself during transcription, serve as size markers. HHRz was prepared as described previously [45] . Lower panel; Western blot targeting C-terminal FLAG tagged ToxN contained within total protein harvested from Pba ToxIN (pMJ4) at different time points, with (+, left) and without (−, right) phage infection. Time 0 indicates a sample taken immediately after infection. Total protein from Pba ToxIN (pMJ4) (−) serves as positive control. (B) Infection with escape phage ΦTE-F. Levels of ToxI were determined by S1-assay (upper) as described in (A) with and without infection. ToxN levels were estimated by Western blotting (lower) as described in (A). (C) Expression of the ΦTE-F ToxI locus. An S1-nuclease assay targeting ToxI was performed on total RNA of Pba (pBR322) at different times during ΦTE-F infection. Pba ToxIN (pMJ4) and DH5α serve as positive and negative controls, respectively.
    E Coli Strain Dh5α, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 48 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Viral Evasion of a Bacterial Suicide System by RNA-Based Molecular Mimicry Enables Infectious Altruism"

    Article Title: Viral Evasion of a Bacterial Suicide System by RNA-Based Molecular Mimicry Enables Infectious Altruism

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1003023

    ΦTE-F expresses ToxI RNA during infection. (A) Upper panel; an S1-nuclease protection assay was used to detect ToxI levels from a ToxIN plasmid during ΦTE wt infection, using an antisense probe against the full 5.5 repeat ToxI sequence [46] . The antisense ToxI-probe was first hybridised to 10 µg of total RNA prepared from Pba ToxIN (pMJ4) at different times after ΦTE infection, and then followed by S1-nuclease treatment. Numbers (+) indicate the time (min) after infection or (−) without the addition of phage. Pba ToxIN-FS (pTA47) and Pba serve as positive and negative controls, respectively. A non-hybridized S1-digested probe (+S1) serves as a further negative control. DH5α 1.5 repeats (pTA96), a non-S1 digested probe (−S1) and an in vitro transcribed Hammerhead ribozyme (HHRz), which cleaves itself during transcription, serve as size markers. HHRz was prepared as described previously [45] . Lower panel; Western blot targeting C-terminal FLAG tagged ToxN contained within total protein harvested from Pba ToxIN (pMJ4) at different time points, with (+, left) and without (−, right) phage infection. Time 0 indicates a sample taken immediately after infection. Total protein from Pba ToxIN (pMJ4) (−) serves as positive control. (B) Infection with escape phage ΦTE-F. Levels of ToxI were determined by S1-assay (upper) as described in (A) with and without infection. ToxN levels were estimated by Western blotting (lower) as described in (A). (C) Expression of the ΦTE-F ToxI locus. An S1-nuclease assay targeting ToxI was performed on total RNA of Pba (pBR322) at different times during ΦTE-F infection. Pba ToxIN (pMJ4) and DH5α serve as positive and negative controls, respectively.
    Figure Legend Snippet: ΦTE-F expresses ToxI RNA during infection. (A) Upper panel; an S1-nuclease protection assay was used to detect ToxI levels from a ToxIN plasmid during ΦTE wt infection, using an antisense probe against the full 5.5 repeat ToxI sequence [46] . The antisense ToxI-probe was first hybridised to 10 µg of total RNA prepared from Pba ToxIN (pMJ4) at different times after ΦTE infection, and then followed by S1-nuclease treatment. Numbers (+) indicate the time (min) after infection or (−) without the addition of phage. Pba ToxIN-FS (pTA47) and Pba serve as positive and negative controls, respectively. A non-hybridized S1-digested probe (+S1) serves as a further negative control. DH5α 1.5 repeats (pTA96), a non-S1 digested probe (−S1) and an in vitro transcribed Hammerhead ribozyme (HHRz), which cleaves itself during transcription, serve as size markers. HHRz was prepared as described previously [45] . Lower panel; Western blot targeting C-terminal FLAG tagged ToxN contained within total protein harvested from Pba ToxIN (pMJ4) at different time points, with (+, left) and without (−, right) phage infection. Time 0 indicates a sample taken immediately after infection. Total protein from Pba ToxIN (pMJ4) (−) serves as positive control. (B) Infection with escape phage ΦTE-F. Levels of ToxI were determined by S1-assay (upper) as described in (A) with and without infection. ToxN levels were estimated by Western blotting (lower) as described in (A). (C) Expression of the ΦTE-F ToxI locus. An S1-nuclease assay targeting ToxI was performed on total RNA of Pba (pBR322) at different times during ΦTE-F infection. Pba ToxIN (pMJ4) and DH5α serve as positive and negative controls, respectively.

    Techniques Used: Infection, Plasmid Preparation, Sequencing, Negative Control, In Vitro, Western Blot, Positive Control, Expressing, Nuclease Assay

    Analysis of pseudo-ToxI as a potential antitoxin. (A) Alignment of the pseudo-ToxI and ToxI RNA sequences. Pseudo-ToxI nucleotides are coloured to match (B) and (C), with the green and purple bases denoting the 5′ and 3′ ends of a single pseudoknot, respectively. Mutated nucleotides in pseudo-ToxI are coloured orange and numbered according to their grouping, whilst the asterisk indicates the additional 3′ nucleotide. The dotted line connecting the U in group 3 indicates the uracil that is deleted in the case of expanded repeats with 2T sequences rather than 3T. (B) Schematic of the ToxI pseudoknot. Each position containing a mutation in the pseudo-ToxI RNA has been bracketed, with the ToxI base separated from the pseudo-ToxI base by a ‘/’. The mutations have been grouped 1–5, according to position, and highlighted in orange, with the 5′ and 3′ termini in green and violet, respectively. Indels, such as U17 that is deleted in some pseudo-ToxI repeats, and the additional A* inserted in all, have been bordered by a dashed line. Base interactions are indicated by black lines, and duplex and triplex base-interactions are bordered in grey. (C) Detail of the ToxIN trimer with each pseudoknot shown either in blue, purple or beige. Each ToxN monomer is shown as a grey surface. The blue pseudoknot is oriented relative to (B). The positions of mutation groups are shown, with the group number encircled in the same colour as the corresponding pseudoknot. The additional nucleotide of group 5 is not visible as this was not in the original solved ToxIN structure. PDB: 2XDB. (D) Pseudo-ToxI cannot protect from ToxN in an over-expression assay. Protection assays were conducted as per Materials and Methods using strains of E. coli DH5α carrying both pTA49 (inducible ToxN) and a second inducible antitoxin vector as shown, including use of pTA100 as a vector-only control, “vector”. Error bars indicate the standard deviation of triplicate data. (E) Protection assays using mutants of ToxI carried out as in (D) with the antitoxin mutations in each construct numbered as per (B). (F) Protection assays carried out as in (D), testing the full escape loci of ΦTE wt, ΦTE-A and ΦTE-F with full ToxI as a positive control. Under these conditions, there was sufficient antitoxin present to inhibit induced ToxN even without specific induction of the ToxI and ΦTE-F constructs.
    Figure Legend Snippet: Analysis of pseudo-ToxI as a potential antitoxin. (A) Alignment of the pseudo-ToxI and ToxI RNA sequences. Pseudo-ToxI nucleotides are coloured to match (B) and (C), with the green and purple bases denoting the 5′ and 3′ ends of a single pseudoknot, respectively. Mutated nucleotides in pseudo-ToxI are coloured orange and numbered according to their grouping, whilst the asterisk indicates the additional 3′ nucleotide. The dotted line connecting the U in group 3 indicates the uracil that is deleted in the case of expanded repeats with 2T sequences rather than 3T. (B) Schematic of the ToxI pseudoknot. Each position containing a mutation in the pseudo-ToxI RNA has been bracketed, with the ToxI base separated from the pseudo-ToxI base by a ‘/’. The mutations have been grouped 1–5, according to position, and highlighted in orange, with the 5′ and 3′ termini in green and violet, respectively. Indels, such as U17 that is deleted in some pseudo-ToxI repeats, and the additional A* inserted in all, have been bordered by a dashed line. Base interactions are indicated by black lines, and duplex and triplex base-interactions are bordered in grey. (C) Detail of the ToxIN trimer with each pseudoknot shown either in blue, purple or beige. Each ToxN monomer is shown as a grey surface. The blue pseudoknot is oriented relative to (B). The positions of mutation groups are shown, with the group number encircled in the same colour as the corresponding pseudoknot. The additional nucleotide of group 5 is not visible as this was not in the original solved ToxIN structure. PDB: 2XDB. (D) Pseudo-ToxI cannot protect from ToxN in an over-expression assay. Protection assays were conducted as per Materials and Methods using strains of E. coli DH5α carrying both pTA49 (inducible ToxN) and a second inducible antitoxin vector as shown, including use of pTA100 as a vector-only control, “vector”. Error bars indicate the standard deviation of triplicate data. (E) Protection assays using mutants of ToxI carried out as in (D) with the antitoxin mutations in each construct numbered as per (B). (F) Protection assays carried out as in (D), testing the full escape loci of ΦTE wt, ΦTE-A and ΦTE-F with full ToxI as a positive control. Under these conditions, there was sufficient antitoxin present to inhibit induced ToxN even without specific induction of the ToxI and ΦTE-F constructs.

    Techniques Used: Mutagenesis, Over Expression, Plasmid Preparation, Standard Deviation, Construct, Positive Control

    2) Product Images from "Molecular Characterization of Inulosucrase from Leuconostoc citreum: a Fructosyltransferase within a Glucosyltransferase"

    Article Title: Molecular Characterization of Inulosucrase from Leuconostoc citreum: a Fructosyltransferase within a Glucosyltransferase

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.185.12.3606-3612.2003

    Representation of three versions of the inulosucrase proteins that were expressed in E. coli DH5α. EIS is the complete enzyme; EIS2 is the enzyme with a deletion of the high-homology C-terminal alternansucrase (ASR), and EIS3 is the enzyme with a deletion of the same region of EIS2 plus a transition region.
    Figure Legend Snippet: Representation of three versions of the inulosucrase proteins that were expressed in E. coli DH5α. EIS is the complete enzyme; EIS2 is the enzyme with a deletion of the high-homology C-terminal alternansucrase (ASR), and EIS3 is the enzyme with a deletion of the same region of EIS2 plus a transition region.

    Techniques Used: Impedance Spectroscopy

    3) Product Images from "Streptococcus pneumoniae DivIVA: Localization and Interactions in a MinCD-Free Context ▿ DivIVA: Localization and Interactions in a MinCD-Free Context ▿ †"

    Article Title: Streptococcus pneumoniae DivIVA: Localization and Interactions in a MinCD-Free Context ▿ DivIVA: Localization and Interactions in a MinCD-Free Context ▿ †

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.01168-06

    Immunoprecipitation to determine the specificity of DivIVA interactions. (A) Immunoprecipitation of GFP-DivIVA-cI-Spo0J complex (lane 1), GFP-Spo0J-cI-DivIVA complex (lane 2), GFP-FtsZ-cI-DivIVA complex (lane 3), and GFP-DivIVA-cI-FtsZ complex (lane 4). (B) Immunoprecipitation of cI-DivIVA-GFP-PrfA complex (lane 1). Protein extracts were immunoprecipitated with anti-GFP antibodies and were detected with anti-cI rabbit polyclonal antibodies by Western blotting as described in Materials and Methods. Lane M contained molecular weight markers. In panel B lane 2 contained crude extract of E. coli strain DH5α expressing cI-DivIVA, detected with anti-cI antibodies.
    Figure Legend Snippet: Immunoprecipitation to determine the specificity of DivIVA interactions. (A) Immunoprecipitation of GFP-DivIVA-cI-Spo0J complex (lane 1), GFP-Spo0J-cI-DivIVA complex (lane 2), GFP-FtsZ-cI-DivIVA complex (lane 3), and GFP-DivIVA-cI-FtsZ complex (lane 4). (B) Immunoprecipitation of cI-DivIVA-GFP-PrfA complex (lane 1). Protein extracts were immunoprecipitated with anti-GFP antibodies and were detected with anti-cI rabbit polyclonal antibodies by Western blotting as described in Materials and Methods. Lane M contained molecular weight markers. In panel B lane 2 contained crude extract of E. coli strain DH5α expressing cI-DivIVA, detected with anti-cI antibodies.

    Techniques Used: Immunoprecipitation, Western Blot, Molecular Weight, Expressing

    4) Product Images from "An engineered autotransporter-based surface expression vector enables efficient display of Affibody molecules on OmpT-negative E. coli as well as protease-mediated secretion in OmpT-positive strains"

    Article Title: An engineered autotransporter-based surface expression vector enables efficient display of Affibody molecules on OmpT-negative E. coli as well as protease-mediated secretion in OmpT-positive strains

    Journal: Microbial Cell Factories

    doi: 10.1186/s12934-014-0179-z

    Evaluation of OmpT-mediated release and small-scale purification of displayed Affibody molecule. A . Flow-cytometric analysis for comparison of surface expression of recombinant proteins between p AraBAD -Z IgG -EC in OmpT-negative E. coli BL21 and p AraBAD -Z IgG -EC in OmpT-positive E. coli DH5α. B . SDS-PAGE showing IMAC-purified supernatants from p AraBAD -Z IgG -EC in OmpT-negative E. coli BL21 and p AraBAD -Z IgG -EC in OmpT-positive E. coli DH5α. Supernatants from non-induced samples, indicated with (−), were included as controls. C . Mass spectrum from ESI-MS analysis of IMAC-purified supernatants from p AraBAD -Z IgG -EC in OmpT-positive E. coli DH5α. Theoretical molecular weight of the OmpT-cleaved Z IgG -His 6 is 9712 Da. D . SPR-based biosensor analysis on the IMAC-purified Z IgG -His 6 . Response units (RU) on the y-axis and time on the x-axis. Representative sensorgrams from injection of Z IgG -His 6 at three different concentrations over human IgG immobilized on the chip surface. Injections were performed in duplicates.
    Figure Legend Snippet: Evaluation of OmpT-mediated release and small-scale purification of displayed Affibody molecule. A . Flow-cytometric analysis for comparison of surface expression of recombinant proteins between p AraBAD -Z IgG -EC in OmpT-negative E. coli BL21 and p AraBAD -Z IgG -EC in OmpT-positive E. coli DH5α. B . SDS-PAGE showing IMAC-purified supernatants from p AraBAD -Z IgG -EC in OmpT-negative E. coli BL21 and p AraBAD -Z IgG -EC in OmpT-positive E. coli DH5α. Supernatants from non-induced samples, indicated with (−), were included as controls. C . Mass spectrum from ESI-MS analysis of IMAC-purified supernatants from p AraBAD -Z IgG -EC in OmpT-positive E. coli DH5α. Theoretical molecular weight of the OmpT-cleaved Z IgG -His 6 is 9712 Da. D . SPR-based biosensor analysis on the IMAC-purified Z IgG -His 6 . Response units (RU) on the y-axis and time on the x-axis. Representative sensorgrams from injection of Z IgG -His 6 at three different concentrations over human IgG immobilized on the chip surface. Injections were performed in duplicates.

    Techniques Used: Purification, Flow Cytometry, Expressing, Recombinant, SDS Page, Mass Spectrometry, Molecular Weight, SPR Assay, Injection, Chromatin Immunoprecipitation

    5) Product Images from "Identification and classification of bacterial Type III toxin-antitoxin systems encoded in chromosomal and plasmid genomes"

    Article Title: Identification and classification of bacterial Type III toxin-antitoxin systems encoded in chromosomal and plasmid genomes

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gks231

    Protection of E. coli DH5α from Type III toxins by cognate antitoxins. Protection assays were performed as described in Materials and Methods. Results for the toxIN system of P. atrosepticum have been published previously ( 9 ); data from a single toxIN experiment is included for illustrative purposes. Of the four new loci tested, all toxin genes reduced viability of the host E. coli , which could then be restored by the full cognate antitoxin. Data shown are the mean values from triplicate experiments, with standard deviations represented by error bars.
    Figure Legend Snippet: Protection of E. coli DH5α from Type III toxins by cognate antitoxins. Protection assays were performed as described in Materials and Methods. Results for the toxIN system of P. atrosepticum have been published previously ( 9 ); data from a single toxIN experiment is included for illustrative purposes. Of the four new loci tested, all toxin genes reduced viability of the host E. coli , which could then be restored by the full cognate antitoxin. Data shown are the mean values from triplicate experiments, with standard deviations represented by error bars.

    Techniques Used:

    6) Product Images from "Biochemical Characterization and Physiological Properties of Escherichia coli UDP-N-Acetylmuramate:l-Alanyl-?-d-Glutamyl-meso- Diaminopimelate Ligase ▿"

    Article Title: Biochemical Characterization and Physiological Properties of Escherichia coli UDP-N-Acetylmuramate:l-Alanyl-?-d-Glutamyl-meso- Diaminopimelate Ligase ▿

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.00087-07

    Overproduction and purification of the Mpl protein (His 6 -tagged form), as determined by SDS-PAGE. Lane MW, molecular mass markers; lane A, total extract from DH5α cells; lane B, total extract from IPTG-induced DH5α cells carrying the pMLD131
    Figure Legend Snippet: Overproduction and purification of the Mpl protein (His 6 -tagged form), as determined by SDS-PAGE. Lane MW, molecular mass markers; lane A, total extract from DH5α cells; lane B, total extract from IPTG-induced DH5α cells carrying the pMLD131

    Techniques Used: Purification, SDS Page

    7) Product Images from "Outer Surface Protein B Is Critical for Borrelia burgdorferi Adherence and Survival within Ixodes Ticks"

    Article Title: Outer Surface Protein B Is Critical for Borrelia burgdorferi Adherence and Survival within Ixodes Ticks

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.0030033

    Characterization of the ospB Mutant and the OspB Complemented Mutant (A) Strategy for the inactivation of the ospB gene and for the generation of the OspB complemented mutant is shown. The top diagram represents the suicide vector pXLF11303 used for transformation into B. burgdorferi B31. Due to homologous recombination (double crossover) between the sequences of pXLF11303 and the native ospAB locus in B. burgdorferi B31, the Δ ospB::kan fragment is inserted into the genome, resulting in the generation of the ospB mutant ( ospB − ). For the generation of the OspB complemented mutant, the P ospAB and ospB gene were PCR-amplified from B. burgdorferi B31 total genomic DNA and cloned into the multiple cloning site (MCS) of the shuttle vector pKFSS1 [ 40 ], resulting in the construct pFGN1 (bottom diagram). The relevant structures of the plasmid and genome are shown. Arrows represent positions of the oligonucleotides used for the PCR analysis. Primer names are retained as shown in Table 1 . Vertical lines represent the restriction enzyme sites. E, EcoRI; N, NotI; and P, PstI. (B) PCR analyses of genomic DNA isolated from spirochetes were performed to confirm the inactivated ospB locus. wt, the wild-type isolate; ospB, the ospB mutant; ospB − , recovered-spirochetes recovered from mice infected with the ospB mutant; ospB − /pFGN1, the OspB complemented mutant; M, 1-kb DNA ladder; C in (B and E) refers to PCR reactions without template DNA. (C) RT-PCR analysis of ospA, ospB, and flaB transcripts in spirochetes grown in BSK-H media. Shown is a gel image of RT-PCR reactions performed with (+RT) and without (−RT) reverse transcriptase. C refers to RT-PCR reactions without cDNA. (D) SDS-PAGE (top) and immunoblot (bottom) analysis of whole-cell lysates of spirochetes. Arrows indicate the expression of OspA, OspB, and FlaB. Immunoblotting analysis performed with monoclonal antibodies directed against OspB (mAb B22J), OspA (C3.78), and FlaB (H9729) were reported previously [ 22 , 50 ]. (E) PCR analyses of genomic DNA to confirm the OspB complementation. (F) Restriction digestion of pFGN1 plasmid recovered from the OspB complemented mutant (pFGN1-recovered) and plasmid controls pFGN1 and pKFSS1. Whole-cell lysate from the OspB complemented mutant was transformed into E. coli DH5α-competent cells and transformed clones were selected in the presence of spectinomycin (50 μg/ml). Plasmids isolated from E. coli cells were digested with NotI or EcoRI and PstI. Expected sizes of restrictive fragments are (i) 5,638 bp and 1,906 bp for NotI digestion of pFGN1; (ii) 5,638 bp and 453 bp for NotI digestion of pKFSS1; (iii) 3,840 bp, 2,413 bp, and 1,291 bp for EcoRI - PstI digestion of pFGN1; and (iv) 3,635 bp, 2,413 bp, and 43 bp for EcoRI - PstI digestion of pKFSS1.
    Figure Legend Snippet: Characterization of the ospB Mutant and the OspB Complemented Mutant (A) Strategy for the inactivation of the ospB gene and for the generation of the OspB complemented mutant is shown. The top diagram represents the suicide vector pXLF11303 used for transformation into B. burgdorferi B31. Due to homologous recombination (double crossover) between the sequences of pXLF11303 and the native ospAB locus in B. burgdorferi B31, the Δ ospB::kan fragment is inserted into the genome, resulting in the generation of the ospB mutant ( ospB − ). For the generation of the OspB complemented mutant, the P ospAB and ospB gene were PCR-amplified from B. burgdorferi B31 total genomic DNA and cloned into the multiple cloning site (MCS) of the shuttle vector pKFSS1 [ 40 ], resulting in the construct pFGN1 (bottom diagram). The relevant structures of the plasmid and genome are shown. Arrows represent positions of the oligonucleotides used for the PCR analysis. Primer names are retained as shown in Table 1 . Vertical lines represent the restriction enzyme sites. E, EcoRI; N, NotI; and P, PstI. (B) PCR analyses of genomic DNA isolated from spirochetes were performed to confirm the inactivated ospB locus. wt, the wild-type isolate; ospB, the ospB mutant; ospB − , recovered-spirochetes recovered from mice infected with the ospB mutant; ospB − /pFGN1, the OspB complemented mutant; M, 1-kb DNA ladder; C in (B and E) refers to PCR reactions without template DNA. (C) RT-PCR analysis of ospA, ospB, and flaB transcripts in spirochetes grown in BSK-H media. Shown is a gel image of RT-PCR reactions performed with (+RT) and without (−RT) reverse transcriptase. C refers to RT-PCR reactions without cDNA. (D) SDS-PAGE (top) and immunoblot (bottom) analysis of whole-cell lysates of spirochetes. Arrows indicate the expression of OspA, OspB, and FlaB. Immunoblotting analysis performed with monoclonal antibodies directed against OspB (mAb B22J), OspA (C3.78), and FlaB (H9729) were reported previously [ 22 , 50 ]. (E) PCR analyses of genomic DNA to confirm the OspB complementation. (F) Restriction digestion of pFGN1 plasmid recovered from the OspB complemented mutant (pFGN1-recovered) and plasmid controls pFGN1 and pKFSS1. Whole-cell lysate from the OspB complemented mutant was transformed into E. coli DH5α-competent cells and transformed clones were selected in the presence of spectinomycin (50 μg/ml). Plasmids isolated from E. coli cells were digested with NotI or EcoRI and PstI. Expected sizes of restrictive fragments are (i) 5,638 bp and 1,906 bp for NotI digestion of pFGN1; (ii) 5,638 bp and 453 bp for NotI digestion of pKFSS1; (iii) 3,840 bp, 2,413 bp, and 1,291 bp for EcoRI - PstI digestion of pFGN1; and (iv) 3,635 bp, 2,413 bp, and 43 bp for EcoRI - PstI digestion of pKFSS1.

    Techniques Used: Mutagenesis, Plasmid Preparation, Transformation Assay, Homologous Recombination, Polymerase Chain Reaction, Amplification, Clone Assay, Construct, Isolation, Mouse Assay, Infection, Reverse Transcription Polymerase Chain Reaction, SDS Page, Expressing

    8) Product Images from "Substrate Profiling of Tobacco Etch Virus Protease Using a Novel Fluorescence-Assisted Whole-Cell Assay"

    Article Title: Substrate Profiling of Tobacco Etch Virus Protease Using a Novel Fluorescence-Assisted Whole-Cell Assay

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0016136

    Flow cytometry analysis of individual clones that emerged through screening of library 1 and 2. Each histogram corresponds to a pure culture of DH5α cells coexpressing TEVp and a reporter construct containing a unique substrate peptide, 2.5 h after induction (0.1 mM IPTG, 0.2% arabinose). The different peptides analyzed are indicated in the figure.
    Figure Legend Snippet: Flow cytometry analysis of individual clones that emerged through screening of library 1 and 2. Each histogram corresponds to a pure culture of DH5α cells coexpressing TEVp and a reporter construct containing a unique substrate peptide, 2.5 h after induction (0.1 mM IPTG, 0.2% arabinose). The different peptides analyzed are indicated in the figure.

    Techniques Used: Flow Cytometry, Cytometry, Clone Assay, Construct

    Screening of TEVp substrate libraries. Three different combinatorial substrate libraries were created, using NNK degenerate codons for randomization of position P6, P3, P1 (Lib1); P6, P3, P1, P1′ (Lib2); or P5, P4, P2, P1′ (Lib3) within the cognate TEVp substrate peptide (ENLYFQG). The libraries were then screened for optimal TEVp substrates. (A) Pre-sorting procedure to eliminate false positive clones from the libraries: DH5α/pMal-TEV2/pGFP-Lib1-ssrA NY , DH5α/pMal-TEV2/pGFP-Lib2-ssrA NY or DH5α/pMal-TEV2/pGFP-Lib3-ssrA NY cells expressing the substrate libraries alone (i.e., TEVp expression not induced) were analyzed on a flow cytometer and non-fluorescent cells were collected through sorting. Here, the original non-sorted population from library 2 and the corresponding “false-positive depleted” library (after two rounds of sorting) are represented by the histograms in purple and jade, respectively. Library 1 and 3 exhibited the same appearance as library 2 (data not shown). (B) Enrichment-progress when screening the libraries for functional TEVp substrates: The false-positive depleted libraries (see Figure 2A ), now coexpressing TEVp and the substrate libraries, were subjected to quantitative flow cytometry analysis, and highly fluorescent cells were collected. The populations from the false-positive depleted library 2, before (jade), after the first (black) and second round of sorting (green) are shown. All three libraries had similar appearance (data not shown).
    Figure Legend Snippet: Screening of TEVp substrate libraries. Three different combinatorial substrate libraries were created, using NNK degenerate codons for randomization of position P6, P3, P1 (Lib1); P6, P3, P1, P1′ (Lib2); or P5, P4, P2, P1′ (Lib3) within the cognate TEVp substrate peptide (ENLYFQG). The libraries were then screened for optimal TEVp substrates. (A) Pre-sorting procedure to eliminate false positive clones from the libraries: DH5α/pMal-TEV2/pGFP-Lib1-ssrA NY , DH5α/pMal-TEV2/pGFP-Lib2-ssrA NY or DH5α/pMal-TEV2/pGFP-Lib3-ssrA NY cells expressing the substrate libraries alone (i.e., TEVp expression not induced) were analyzed on a flow cytometer and non-fluorescent cells were collected through sorting. Here, the original non-sorted population from library 2 and the corresponding “false-positive depleted” library (after two rounds of sorting) are represented by the histograms in purple and jade, respectively. Library 1 and 3 exhibited the same appearance as library 2 (data not shown). (B) Enrichment-progress when screening the libraries for functional TEVp substrates: The false-positive depleted libraries (see Figure 2A ), now coexpressing TEVp and the substrate libraries, were subjected to quantitative flow cytometry analysis, and highly fluorescent cells were collected. The populations from the false-positive depleted library 2, before (jade), after the first (black) and second round of sorting (green) are shown. All three libraries had similar appearance (data not shown).

    Techniques Used: Clone Assay, Expressing, Flow Cytometry, Cytometry, Functional Assay

    9) Product Images from "Inhibition of FtsZ polymerization by SulA, an inhibitor of septation in Escherichia coli"

    Article Title: Inhibition of FtsZ polymerization by SulA, an inhibitor of septation in Escherichia coli

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi:

    Cell morphology and Z ring formation after induction of MalE-SulA fusions. ( A ) Cultures of DH5α (pJC93) or (pJC94) were grown in L broth with glucose. Arabinose was added at 0.05% and incubation was continued for 1 hr. Samples were taken and cells were immunostained with antibodies to FtsZ. The arrows indicate cells that have constrictions but lack Z rings. (×1,000.) ( B ) The induction of the MalE-SulA fusions monitored by immunoblot analysis. Samples from the cultures in A as well as several additional cultures with less arabinose were taken for immunoblotting. The samples were separated by SDS/PAGE, transferred to nitrocellulose, and immunostained with antibodies against FtsZ and the MalE-SulA fusion. The bands corresponding to FtsZ and the MalE-SulA fusions are indicated.
    Figure Legend Snippet: Cell morphology and Z ring formation after induction of MalE-SulA fusions. ( A ) Cultures of DH5α (pJC93) or (pJC94) were grown in L broth with glucose. Arabinose was added at 0.05% and incubation was continued for 1 hr. Samples were taken and cells were immunostained with antibodies to FtsZ. The arrows indicate cells that have constrictions but lack Z rings. (×1,000.) ( B ) The induction of the MalE-SulA fusions monitored by immunoblot analysis. Samples from the cultures in A as well as several additional cultures with less arabinose were taken for immunoblotting. The samples were separated by SDS/PAGE, transferred to nitrocellulose, and immunostained with antibodies against FtsZ and the MalE-SulA fusion. The bands corresponding to FtsZ and the MalE-SulA fusions are indicated.

    Techniques Used: Incubation, SDS Page

    10) Product Images from "Novel Fluorescence-Assisted Whole-Cell Assay for Engineering and Characterization of Proteases and Their Substrates ▿"

    Article Title: Novel Fluorescence-Assisted Whole-Cell Assay for Engineering and Characterization of Proteases and Their Substrates ▿

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.01558-10

    System development and optimizations. Fluorescence intensity of E. coli DH5α cells, harboring expression vectors for TEVp and different reporter constructs, subjected to various induction conditions. (A) DH5α/pMal-TEV2/pGFP-subG-ssrA (where
    Figure Legend Snippet: System development and optimizations. Fluorescence intensity of E. coli DH5α cells, harboring expression vectors for TEVp and different reporter constructs, subjected to various induction conditions. (A) DH5α/pMal-TEV2/pGFP-subG-ssrA (where

    Techniques Used: Fluorescence, Expressing, Construct

    Discrimination between protease substrates and isolation of an efficiently processed peptide from a large background of suboptimal substrates. (A) Fluorescence intensity of DH5α/pMal-TEV2/pGFP-ssrA NY , DH5α/pMal-TEV2/pGFP-subG-ssrA NY , DH5α/pMal-TEV2/pGFP-subP-ssrA
    Figure Legend Snippet: Discrimination between protease substrates and isolation of an efficiently processed peptide from a large background of suboptimal substrates. (A) Fluorescence intensity of DH5α/pMal-TEV2/pGFP-ssrA NY , DH5α/pMal-TEV2/pGFP-subG-ssrA NY , DH5α/pMal-TEV2/pGFP-subP-ssrA

    Techniques Used: Isolation, Fluorescence

    Discrimination of TEVp variants exhibiting different in vivo solubility and isolation of the most soluble one from a large background of a less-soluble TEVp variant. (A) Fluorescence intensity of DH5α/pMal-TEV2/pGFP-ssrA NY , DH5α/pTEV/pGFP-subG-ssrA
    Figure Legend Snippet: Discrimination of TEVp variants exhibiting different in vivo solubility and isolation of the most soluble one from a large background of a less-soluble TEVp variant. (A) Fluorescence intensity of DH5α/pMal-TEV2/pGFP-ssrA NY , DH5α/pTEV/pGFP-subG-ssrA

    Techniques Used: In Vivo, Solubility, Isolation, Variant Assay, Fluorescence

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