att b2 1 sites  (Thermo Fisher)


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    Name:
    Zero Blunt TOPO PCR Cloning Kit for Sequencing
    Description:
    Zero Blunt TOPO PCR Cloning kits for sequencing provide a highly efficient 5 minute cloning reaction for the direct insertion of proofreading polymerase amplified blunt ended PCR products into a plasmid vector for sequencing Each kit uses the pCR 4Blunt TOPO vector with specially designed sequencing primer sites that return more insert sequence and less vector sequence from each reaction These kits include the tools necessary to clone and select recombinant vectors containing your PCR fragment of choice • Fast and easy go from PCR to clones in just 3 steps including a 5 minute cloning reaction • Efficient obtain up to 95 clones with correct insert • Proven enabling reliable performance for over a decade with over 20 000 citations • Simple no ligase post PCR procedures or PCR primers containing specific sequences required • Accurate for use with high fidelity DNA polymerases no need for A overhang on PCR product pCR 4Blunt TOPO Vector Optimized for Sequencing The pCR 4Blunt TOPO vector has a minimized multiple cloning site and thus a shorter distance between the sequencing primer sites and the insert site as little as 33 bp So sequencing reactions produce less vector sequence and more insert sequence The pCR 4Blunt TOPO vector has sites for 4 common sequencing primers M13 forward M13 reverse T7 and T3 The kits include an aliquot of each pCR 4Blunt TOPO Clone Selection and Manipulation The pCR 4Blunt TOPO vector contains both ampicillin and kanamycin resistance markers and a LacZα ccdB gene fusion for positive selection The vector s minimized multiple cloning site still includes flanking EcoRI sites for simplified excision of cloned PCR products and a unique Sse8387I site for generation of nested deletions prior to sequencing T7 and T3 promoters are also present for in vitro transcription Simplified TOPO based Cloning Using TOPO cloning technology there is no need for PCR primers containing specific sequences post PCR procedures vector preparation or other time intensive DNA manipulation steps Just add your PCR reaction straight to the provided topoisomerase charged vector incubate 5 minutes and transform E coli competent cells Efficient Cloning With up to 95 of clones carrying the desired insert you can screen fewer clones which saves time and money The pCR 4Blunt TOPO vector has no overhangs for efficient ligation of PCR products created by proofreading thermostable polymerases that leave blunt ended PCR products The Most Widely Used Cloning Kit When it comes to cloning TOPO cloning technology has been a reliable partner for thousands of scientists for over ten years Fast simple to use and efficient TOPO cloning has been applied to many different vectors for a wide array of applications Kit Options Zero Blunt TOPO PCR Cloning Kits for Sequencing Zero Blunt TOPO PCR Cloning kits for sequencing can be purchased with a variety of competent cells that deliver different advantages depending upon your needs • General cloning TOP10 cells Cat Nos K2875 J10 K2875 20 K2875 40 • High efficiency cloning TOP10 Electrocomp cells Cat Nos K2880 20 K2880 40 • General cloning bacteriophage T1 resistance DH5α T1R Cat No K2895 20 • Fast growth Mach1 T1R chemically competent E coli Cat No K2835 20 • Provide your own cells Cat Nos 450159 and 450031
    Catalog Number:
    450031
    Price:
    None
    Applications:
    Cloning|PCR Cloning
    Category:
    DNA Vectors Clones Purified Nucleic Acids Libraries
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    Structured Review

    Thermo Fisher att b2 1 sites
    Schematic representation of the high-throughput cloning strategy. A. B. subtilis Gateway expression vector pDG148-GW with an inducible P spac promoter. lacI, lac repressor gene; AmpR, kanR, phlR, CmR, genes providing resistance to ampicilin, kanamycin, phleomycin, or chloramphenicol, respectively; ccdB, gene coding for the cytotoxic CcdB protein. B. Full-length coding sequences (CDS) are amplified by nested PCR using a set of CDS-specific (primary) primers and a set of universal (secondary) primers. The resulting PCR product contains the CDS preceded by a synthetic SD sequence (brackets) and a Sap I restriction site (underlined). att B1.1 and att <t>B2.1</t> sites for site-specific recombination are indicated by braces; start and stop codons of the CDS, in italic-bold.
    Zero Blunt TOPO PCR Cloning kits for sequencing provide a highly efficient 5 minute cloning reaction for the direct insertion of proofreading polymerase amplified blunt ended PCR products into a plasmid vector for sequencing Each kit uses the pCR 4Blunt TOPO vector with specially designed sequencing primer sites that return more insert sequence and less vector sequence from each reaction These kits include the tools necessary to clone and select recombinant vectors containing your PCR fragment of choice • Fast and easy go from PCR to clones in just 3 steps including a 5 minute cloning reaction • Efficient obtain up to 95 clones with correct insert • Proven enabling reliable performance for over a decade with over 20 000 citations • Simple no ligase post PCR procedures or PCR primers containing specific sequences required • Accurate for use with high fidelity DNA polymerases no need for A overhang on PCR product pCR 4Blunt TOPO Vector Optimized for Sequencing The pCR 4Blunt TOPO vector has a minimized multiple cloning site and thus a shorter distance between the sequencing primer sites and the insert site as little as 33 bp So sequencing reactions produce less vector sequence and more insert sequence The pCR 4Blunt TOPO vector has sites for 4 common sequencing primers M13 forward M13 reverse T7 and T3 The kits include an aliquot of each pCR 4Blunt TOPO Clone Selection and Manipulation The pCR 4Blunt TOPO vector contains both ampicillin and kanamycin resistance markers and a LacZα ccdB gene fusion for positive selection The vector s minimized multiple cloning site still includes flanking EcoRI sites for simplified excision of cloned PCR products and a unique Sse8387I site for generation of nested deletions prior to sequencing T7 and T3 promoters are also present for in vitro transcription Simplified TOPO based Cloning Using TOPO cloning technology there is no need for PCR primers containing specific sequences post PCR procedures vector preparation or other time intensive DNA manipulation steps Just add your PCR reaction straight to the provided topoisomerase charged vector incubate 5 minutes and transform E coli competent cells Efficient Cloning With up to 95 of clones carrying the desired insert you can screen fewer clones which saves time and money The pCR 4Blunt TOPO vector has no overhangs for efficient ligation of PCR products created by proofreading thermostable polymerases that leave blunt ended PCR products The Most Widely Used Cloning Kit When it comes to cloning TOPO cloning technology has been a reliable partner for thousands of scientists for over ten years Fast simple to use and efficient TOPO cloning has been applied to many different vectors for a wide array of applications Kit Options Zero Blunt TOPO PCR Cloning Kits for Sequencing Zero Blunt TOPO PCR Cloning kits for sequencing can be purchased with a variety of competent cells that deliver different advantages depending upon your needs • General cloning TOP10 cells Cat Nos K2875 J10 K2875 20 K2875 40 • High efficiency cloning TOP10 Electrocomp cells Cat Nos K2880 20 K2880 40 • General cloning bacteriophage T1 resistance DH5α T1R Cat No K2895 20 • Fast growth Mach1 T1R chemically competent E coli Cat No K2835 20 • Provide your own cells Cat Nos 450159 and 450031
    https://www.bioz.com/result/att b2 1 sites/product/Thermo Fisher
    Average 85 stars, based on 13388 article reviews
    Price from $9.99 to $1999.99
    att b2 1 sites - by Bioz Stars, 2020-08
    85/100 stars

    Images

    1) Product Images from "High-Throughput System for the Presentation of Secreted and Surface-Exposed Proteins from Gram-Positive Bacteria in Functional Metagenomics Studies"

    Article Title: High-Throughput System for the Presentation of Secreted and Surface-Exposed Proteins from Gram-Positive Bacteria in Functional Metagenomics Studies

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0065956

    Schematic representation of the high-throughput cloning strategy. A. B. subtilis Gateway expression vector pDG148-GW with an inducible P spac promoter. lacI, lac repressor gene; AmpR, kanR, phlR, CmR, genes providing resistance to ampicilin, kanamycin, phleomycin, or chloramphenicol, respectively; ccdB, gene coding for the cytotoxic CcdB protein. B. Full-length coding sequences (CDS) are amplified by nested PCR using a set of CDS-specific (primary) primers and a set of universal (secondary) primers. The resulting PCR product contains the CDS preceded by a synthetic SD sequence (brackets) and a Sap I restriction site (underlined). att B1.1 and att B2.1 sites for site-specific recombination are indicated by braces; start and stop codons of the CDS, in italic-bold.
    Figure Legend Snippet: Schematic representation of the high-throughput cloning strategy. A. B. subtilis Gateway expression vector pDG148-GW with an inducible P spac promoter. lacI, lac repressor gene; AmpR, kanR, phlR, CmR, genes providing resistance to ampicilin, kanamycin, phleomycin, or chloramphenicol, respectively; ccdB, gene coding for the cytotoxic CcdB protein. B. Full-length coding sequences (CDS) are amplified by nested PCR using a set of CDS-specific (primary) primers and a set of universal (secondary) primers. The resulting PCR product contains the CDS preceded by a synthetic SD sequence (brackets) and a Sap I restriction site (underlined). att B1.1 and att B2.1 sites for site-specific recombination are indicated by braces; start and stop codons of the CDS, in italic-bold.

    Techniques Used: High Throughput Screening Assay, Clone Assay, Expressing, Plasmid Preparation, Amplification, Nested PCR, Polymerase Chain Reaction, Sequencing

    2) Product Images from "Genome-Wide Association Mapping in Dogs Enables Identification of the Homeobox Gene, NKX2-8, as a Genetic Component of Neural Tube Defects in Humans"

    Article Title: Genome-Wide Association Mapping in Dogs Enables Identification of the Homeobox Gene, NKX2-8, as a Genetic Component of Neural Tube Defects in Humans

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1003646

    Chromatograms of NKX2-8 exon 2 sequence where a mutation of G to AA was found in an affected Weimaraner. From left to right: Unaffected Weimaraners have a genotype of “GG”; affected Weimaraners have a genotype of “AA”; carrier Weimaraners have a genotype of “AG”.
    Figure Legend Snippet: Chromatograms of NKX2-8 exon 2 sequence where a mutation of G to AA was found in an affected Weimaraner. From left to right: Unaffected Weimaraners have a genotype of “GG”; affected Weimaraners have a genotype of “AA”; carrier Weimaraners have a genotype of “AG”.

    Techniques Used: Sequencing, Mutagenesis

    Comparison of the protein sequence of NKX2-8 between human, unaffected dog, spinal dysraphism Weimaraner, cat, cow, bat, wild boar, mouse, tree-shrew, chicken and zebra fish. Two functional domains: a homeobox (A) and an NK specific domain (B) are boxed. A truncated protein (arrow), the result of the frameshift mutation (grey shaded) in spinal dysraphism affected Weimaraners, is missing the NK specific domain. Locations for missense variants rs61755040 (asterisk) and rs10135525 (double asterisk), found in human patients with spina bifida, are shaded within the protein sequence. These variants reside within evolutionary conserved domains.
    Figure Legend Snippet: Comparison of the protein sequence of NKX2-8 between human, unaffected dog, spinal dysraphism Weimaraner, cat, cow, bat, wild boar, mouse, tree-shrew, chicken and zebra fish. Two functional domains: a homeobox (A) and an NK specific domain (B) are boxed. A truncated protein (arrow), the result of the frameshift mutation (grey shaded) in spinal dysraphism affected Weimaraners, is missing the NK specific domain. Locations for missense variants rs61755040 (asterisk) and rs10135525 (double asterisk), found in human patients with spina bifida, are shaded within the protein sequence. These variants reside within evolutionary conserved domains.

    Techniques Used: Sequencing, Fluorescence In Situ Hybridization, Functional Assay, Mutagenesis

    Manhattan plots of GWAS results for NTDs in Weimaraners (4 cases, 96 controls; λ = 1.03). A . Raw p-values. Y axis: −log 10 of the raw p-values; X axis: SNPs color coded by chromosome. The lowest p-values are on chromosome 8. B . 100K Max (T) permutation results. Y axis: −log 10 of the permuted p-value; X axis: SNPs color coded by chromosome. The red line denotes genome wide significance (p≤0.05; −log 10≥1.3). C . Chi-square and allele frequencies for affected dogs by Mb on chromosome 8. The interval with the highest chi-square association (χ 2 = 119) and allele frequency = 1 within affected dogs is boxed, defining the critical interval. NKX2-8 is located within this interval.
    Figure Legend Snippet: Manhattan plots of GWAS results for NTDs in Weimaraners (4 cases, 96 controls; λ = 1.03). A . Raw p-values. Y axis: −log 10 of the raw p-values; X axis: SNPs color coded by chromosome. The lowest p-values are on chromosome 8. B . 100K Max (T) permutation results. Y axis: −log 10 of the permuted p-value; X axis: SNPs color coded by chromosome. The red line denotes genome wide significance (p≤0.05; −log 10≥1.3). C . Chi-square and allele frequencies for affected dogs by Mb on chromosome 8. The interval with the highest chi-square association (χ 2 = 119) and allele frequency = 1 within affected dogs is boxed, defining the critical interval. NKX2-8 is located within this interval.

    Techniques Used: GWAS, Genome Wide

    3) Product Images from "Integrated Metagenomic and Physiochemical Analyses to Evaluate the Potential Role of Microbes in the Sand Filter of a Drinking Water Treatment System"

    Article Title: Integrated Metagenomic and Physiochemical Analyses to Evaluate the Potential Role of Microbes in the Sand Filter of a Drinking Water Treatment System

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0061011

    Vertical distribution of 16S rRNA (bacteria and archaea), amoA (bacteria and archaea), and nosZ gene in the sand filter system of the drinking water treatment facility. The results were obtained from real-time PCR assay. Error bars represent standard deviation from three independent experiments.
    Figure Legend Snippet: Vertical distribution of 16S rRNA (bacteria and archaea), amoA (bacteria and archaea), and nosZ gene in the sand filter system of the drinking water treatment facility. The results were obtained from real-time PCR assay. Error bars represent standard deviation from three independent experiments.

    Techniques Used: Real-time Polymerase Chain Reaction, Standard Deviation

    Phylogenetic trees based on the (a) archaeal 16S rRNA and (b) archaeal amoA representative sequences (OTUs, 97% similarity) from the DZhigh sample. The numbers on the branch nodes represent percentage of bootstrap resamplings based on 1000 replicates (only ≥50% are shown). The scale bar indicates the number of nucleotide substitutions per site. The relative abundance of each OTU in each clone library is shown in parentheses.
    Figure Legend Snippet: Phylogenetic trees based on the (a) archaeal 16S rRNA and (b) archaeal amoA representative sequences (OTUs, 97% similarity) from the DZhigh sample. The numbers on the branch nodes represent percentage of bootstrap resamplings based on 1000 replicates (only ≥50% are shown). The scale bar indicates the number of nucleotide substitutions per site. The relative abundance of each OTU in each clone library is shown in parentheses.

    Techniques Used:

    4) Product Images from "Promoter analysis of macrophage- and tick cell-specific differentially expressed Ehrlichia chaffeensis p28-Omp genes"

    Article Title: Promoter analysis of macrophage- and tick cell-specific differentially expressed Ehrlichia chaffeensis p28-Omp genes

    Journal: BMC Microbiology

    doi: 10.1186/1471-2180-9-99

    (A) Green fluorescent protein (GFP) constructs evaluated for the promoter activity of p28-Omp genes 14 and 19 . The pPROBE-NT plasmids containing the promoterless GFP gene (2 and 3) and upstream sequences of genes 14 and 19 in front of the GFP gene (1 and 4, respectively) and a construct containing no promoter sequence were evaluated for GFP expression in E. coli . (B) LacZ constructs evaluated for the promoter activity of p28-Omp genes 14 and 19. The pBlue-TOPO vector containing promoterless lacZ gene (pBlue-TOPO) and upstream sequences of genes 14 and 19 inserted in forward (14-F and 19-F) and reverse orientations (14-R and 19-R) were evaluated for β-galactosidase activity in E. coli . Data are presented with SD values calculated from four independent experiments ( P ≤ 0.001).
    Figure Legend Snippet: (A) Green fluorescent protein (GFP) constructs evaluated for the promoter activity of p28-Omp genes 14 and 19 . The pPROBE-NT plasmids containing the promoterless GFP gene (2 and 3) and upstream sequences of genes 14 and 19 in front of the GFP gene (1 and 4, respectively) and a construct containing no promoter sequence were evaluated for GFP expression in E. coli . (B) LacZ constructs evaluated for the promoter activity of p28-Omp genes 14 and 19. The pBlue-TOPO vector containing promoterless lacZ gene (pBlue-TOPO) and upstream sequences of genes 14 and 19 inserted in forward (14-F and 19-F) and reverse orientations (14-R and 19-R) were evaluated for β-galactosidase activity in E. coli . Data are presented with SD values calculated from four independent experiments ( P ≤ 0.001).

    Techniques Used: Construct, Activity Assay, Sequencing, Expressing, Plasmid Preparation

    5) Product Images from "Signature-Tagged Mutagenesis in a Chicken Infection Model Leads to the Identification of a Novel Avian Pathogenic Escherichia coli Fimbrial Adhesin"

    Article Title: Signature-Tagged Mutagenesis in a Chicken Infection Model Leads to the Identification of a Novel Avian Pathogenic Escherichia coli Fimbrial Adhesin

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0007796

    Physical map showing genomic organization of the 4,975 bp yqi adhesin gene cluster in APEC strain IMT5155. A hypothetical protein preceeds the putative outer membrane usher protein, followed by the putative chaperone and finally the putative adhesin.
    Figure Legend Snippet: Physical map showing genomic organization of the 4,975 bp yqi adhesin gene cluster in APEC strain IMT5155. A hypothetical protein preceeds the putative outer membrane usher protein, followed by the putative chaperone and finally the putative adhesin.

    Techniques Used:

    Bacterial colonization of the chicken lungs 24 h after intra-tracheal infection with 10 6 CFU of bacteria. Differences between IMT5155 and IMT5155Δ yqi were statistically significant with a p
    Figure Legend Snippet: Bacterial colonization of the chicken lungs 24 h after intra-tracheal infection with 10 6 CFU of bacteria. Differences between IMT5155 and IMT5155Δ yqi were statistically significant with a p

    Techniques Used: Infection

    Bacterial adhesion to chicken fibroblast cells 1.5 h and 3 h after infection with an MOI = 100. Differences between IMT5155 and IMT5155Δ yqi were statistically significant with a p
    Figure Legend Snippet: Bacterial adhesion to chicken fibroblast cells 1.5 h and 3 h after infection with an MOI = 100. Differences between IMT5155 and IMT5155Δ yqi were statistically significant with a p

    Techniques Used: Infection

    Expression of the ExPEC adhesin I ( yqi ) gene cluster in vitro . Electron micrographs show negatively stained afimbriate strain E. coli AAEC189 (pKESK: yqi _4975_XB) over-expressed with the yqi adhesin gene cluster at a magnification of 45,000x, 65,000x and 100,000x (A–C), negative control afimbriate strain E. coli AAEC189 (D) and wild type fimbriated E. coli strain IMT5155 (E). The arrows indicate the location of the fimbriae.
    Figure Legend Snippet: Expression of the ExPEC adhesin I ( yqi ) gene cluster in vitro . Electron micrographs show negatively stained afimbriate strain E. coli AAEC189 (pKESK: yqi _4975_XB) over-expressed with the yqi adhesin gene cluster at a magnification of 45,000x, 65,000x and 100,000x (A–C), negative control afimbriate strain E. coli AAEC189 (D) and wild type fimbriated E. coli strain IMT5155 (E). The arrows indicate the location of the fimbriae.

    Techniques Used: Expressing, In Vitro, Staining, Negative Control

    Bacterial re-isolation of IMT5155, IMT5155Δ yqi and IMT11327 from the lungs, spleen, liver, heart, kidneys and brain 24 h after intra-tracheal infection with 10 9 CFU of bacteria (n = 6). Absence of columns indicates that no bacteria were isolated from the organ.
    Figure Legend Snippet: Bacterial re-isolation of IMT5155, IMT5155Δ yqi and IMT11327 from the lungs, spleen, liver, heart, kidneys and brain 24 h after intra-tracheal infection with 10 9 CFU of bacteria (n = 6). Absence of columns indicates that no bacteria were isolated from the organ.

    Techniques Used: Isolation, Infection

    6) Product Images from "Recovery of infectious virus from full-length cowpox virus (CPXV) DNA cloned as a bacterial artificial chromosome (BAC)"

    Article Title: Recovery of infectious virus from full-length cowpox virus (CPXV) DNA cloned as a bacterial artificial chromosome (BAC)

    Journal: Veterinary Research

    doi: 10.1186/1297-9716-42-3

    RFLP analysis of pBR- and pBRf-derived viruses and characterization of mini-F loss in pBR-derived viruses . A. BAC DNA or viral DNA prepared from infected Vero cells was digested with XhoI and separated by 0.8% agarose gel electrophoresis. The lanes contain the DNA's from BAC DNA (pBRf, pBR), viruses reconstituted from cloned DNA (vBRf, vBR) or parental CPXV.BR DNA (BR). Changes of the restriction pattern due to the presence and therefore disruption of TK of absence of mini-F sequences are marked with boxes (see text). B. Southern blot analysis after transfer of the gel shown in (A) and using a DIG labeled TK probe. The changes seen with a DIG labeled TK probe show the expected changes due to the disruption of TK with mini-F sequences (pBRf, vBRf), the introduction of an inverse TK sequence (pBR), and the loss of any bacterial sequences in vBR (see text). Sizes of a molecular weight marker (generuler 1-kb plus DNA ladder, Fermentas) are given. C. Development of GFP fluorescence in plaques following removal of mini-F sequences. Initially, all plaques exhibit GFP fluorescence (plaque in left panels), which is then partially lost (plaque in middle panels) or completely absent (plaque in right panels). Identical plaques were photographed under fluorescent light (upper panels) or using phase contrast (lower panels) with a Zeiss Axiovert microscope and a CCD camera (Zeiss).
    Figure Legend Snippet: RFLP analysis of pBR- and pBRf-derived viruses and characterization of mini-F loss in pBR-derived viruses . A. BAC DNA or viral DNA prepared from infected Vero cells was digested with XhoI and separated by 0.8% agarose gel electrophoresis. The lanes contain the DNA's from BAC DNA (pBRf, pBR), viruses reconstituted from cloned DNA (vBRf, vBR) or parental CPXV.BR DNA (BR). Changes of the restriction pattern due to the presence and therefore disruption of TK of absence of mini-F sequences are marked with boxes (see text). B. Southern blot analysis after transfer of the gel shown in (A) and using a DIG labeled TK probe. The changes seen with a DIG labeled TK probe show the expected changes due to the disruption of TK with mini-F sequences (pBRf, vBRf), the introduction of an inverse TK sequence (pBR), and the loss of any bacterial sequences in vBR (see text). Sizes of a molecular weight marker (generuler 1-kb plus DNA ladder, Fermentas) are given. C. Development of GFP fluorescence in plaques following removal of mini-F sequences. Initially, all plaques exhibit GFP fluorescence (plaque in left panels), which is then partially lost (plaque in middle panels) or completely absent (plaque in right panels). Identical plaques were photographed under fluorescent light (upper panels) or using phase contrast (lower panels) with a Zeiss Axiovert microscope and a CCD camera (Zeiss).

    Techniques Used: Derivative Assay, BAC Assay, Infection, Agarose Gel Electrophoresis, Clone Assay, Southern Blot, Labeling, Sequencing, Molecular Weight, Marker, Fluorescence, Microscopy

    Restriction fragment analysis of full-length BAC clone pBRf . A. BAC DNA was isolated and digested with seven different enzymes (HindIII, KpnI, PstI, SacI, SphI, StuI, XhoI) and separated on a 0.8% agarose gel. Sizes of a molecular weight marker (generuler 1-kb plus DNA ladder, Fermentas) are given. B. Patterns corresponded exactly to the predictions based on the complete CPXV.BR genome (#NC_003663) as shown by in silico digests using VectorNTI.
    Figure Legend Snippet: Restriction fragment analysis of full-length BAC clone pBRf . A. BAC DNA was isolated and digested with seven different enzymes (HindIII, KpnI, PstI, SacI, SphI, StuI, XhoI) and separated on a 0.8% agarose gel. Sizes of a molecular weight marker (generuler 1-kb plus DNA ladder, Fermentas) are given. B. Patterns corresponded exactly to the predictions based on the complete CPXV.BR genome (#NC_003663) as shown by in silico digests using VectorNTI.

    Techniques Used: BAC Assay, Isolation, Agarose Gel Electrophoresis, Molecular Weight, Marker, In Silico

    Schematic presentation of rationale for cloning of cowpox virus infectious clone . A. Shown is a schematic of the full-length CPXV.BR genome and the TK locus in greater detail. In a first step, the viral TK was amplified by PCR, cloned into the pCRII vector, which was then used for an inverse PCR to introduce an Fse I restriction site into TK. Mini-F vector sequences containing gfp under the control of the late 4B promoter were finally inserted into the singular Fse I site to obtain transfer plasmid #7. B. Shown are the constructs and the strategy used to obtain recombinant virus BR.TK- and the full-length CPXV.BR BAC pBRf. Recombinant BR.TK- was used to infect Vero cells. Infection resulted in the formation of replication intermediates, concatemers, some of which were circularized. Viral DNA extracted from cells infected with BR.TK- was electroporated into E. coli DH10B giving rise to pBRf. The full-length CPXV BAC clone was ultimately transfected into Vero cells using FWPV as a helper. Scale bars indicate the sizes of the molecules. Abbreviations: ITR, inverted terminal repeats; cat , chloramphenicol resistance gene; gfp , green fluorescent protein gene: loxP , loxP sites; H,
    Figure Legend Snippet: Schematic presentation of rationale for cloning of cowpox virus infectious clone . A. Shown is a schematic of the full-length CPXV.BR genome and the TK locus in greater detail. In a first step, the viral TK was amplified by PCR, cloned into the pCRII vector, which was then used for an inverse PCR to introduce an Fse I restriction site into TK. Mini-F vector sequences containing gfp under the control of the late 4B promoter were finally inserted into the singular Fse I site to obtain transfer plasmid #7. B. Shown are the constructs and the strategy used to obtain recombinant virus BR.TK- and the full-length CPXV.BR BAC pBRf. Recombinant BR.TK- was used to infect Vero cells. Infection resulted in the formation of replication intermediates, concatemers, some of which were circularized. Viral DNA extracted from cells infected with BR.TK- was electroporated into E. coli DH10B giving rise to pBRf. The full-length CPXV BAC clone was ultimately transfected into Vero cells using FWPV as a helper. Scale bars indicate the sizes of the molecules. Abbreviations: ITR, inverted terminal repeats; cat , chloramphenicol resistance gene; gfp , green fluorescent protein gene: loxP , loxP sites; H, "head", T, "tail" orientation of the ITR present in the replicative intermediates.

    Techniques Used: Clone Assay, Amplification, Polymerase Chain Reaction, Plasmid Preparation, Inverse PCR, Introduce, Construct, Recombinant, BAC Assay, Infection, Transfection

    7) Product Images from "Host–Pathogen Coevolution: The Selective Advantage of Bacillus thuringiensis Virulence and Its Cry Toxin Genes"

    Article Title: Host–Pathogen Coevolution: The Selective Advantage of Bacillus thuringiensis Virulence and Its Cry Toxin Genes

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.1002169

    Virulence of BT-679 pathogens with or without nematocidal toxin genes. Mean virulence of plasmid-lacking BT-679 (Cry-) with reintroduced Cry14Aa1 (+14) or Cry21Aa2 (+21; left panel) or two concentrations of Cry21Aa2-expressing E . coli (+EC21_low, +EC21_high; right panel). Cry+, toxin gene plasmid-bearing BT-679; Cry-_0, empty vector control for BT-679; EC0, empty vector control for E . coli . The data is provided in S6 Data .
    Figure Legend Snippet: Virulence of BT-679 pathogens with or without nematocidal toxin genes. Mean virulence of plasmid-lacking BT-679 (Cry-) with reintroduced Cry14Aa1 (+14) or Cry21Aa2 (+21; left panel) or two concentrations of Cry21Aa2-expressing E . coli (+EC21_low, +EC21_high; right panel). Cry+, toxin gene plasmid-bearing BT-679; Cry-_0, empty vector control for BT-679; EC0, empty vector control for E . coli . The data is provided in S6 Data .

    Techniques Used: Plasmid Preparation, Expressing

    Frequency of BT-679 toxin genes cry21Aa2 and cry35Aa4 among the evolved replicate populations. The different shades of blue indicate alternative combinations of toxin genes present, as indicated. The toxin genes were all restricted to evolved clones of the BT-679 background (i.e., horizontal transfer was not detected). The top two rows refer to the coevolved, the middle two rows to one-sided adapted, and the bottom two rows to the control evolved replicate populations. Replicate populations are given along the horizontal axis. Data is shown for both transfer 12 and 20 and a total of 55 replicate populations. Crosses indicate extinction of replicates and
    Figure Legend Snippet: Frequency of BT-679 toxin genes cry21Aa2 and cry35Aa4 among the evolved replicate populations. The different shades of blue indicate alternative combinations of toxin genes present, as indicated. The toxin genes were all restricted to evolved clones of the BT-679 background (i.e., horizontal transfer was not detected). The top two rows refer to the coevolved, the middle two rows to one-sided adapted, and the bottom two rows to the control evolved replicate populations. Replicate populations are given along the horizontal axis. Data is shown for both transfer 12 and 20 and a total of 55 replicate populations. Crosses indicate extinction of replicates and "miss" that genetic material for the population was unavailable. The original data is shown in S4 Data .

    Techniques Used: Clone Assay

    8) Product Images from "Immunomodulator-Based Enhancement of Anti Smallpox Immune Responses"

    Article Title: Immunomodulator-Based Enhancement of Anti Smallpox Immune Responses

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0123113

    A27L-specific cell-mediated immune response against recombinant VVWR determined by ELISPOT analysis. (A) Each group consisted of four female Balb/C mice immunized three times, two weeks apart via intramuscular injection as follows: Naïve (negative control), pVax1 (empty vector backbone control), pA27L (VVWR envelope protein), pA27L+I (VVWR envelope protein + imiquimod), pA27L + R (VVWR envelope protein + resiquimod), pA27LOPT (optimized sequence of the A27L gene) and pA27LOPT+I (the optimized sequence of A27L + imiquimod). (B) Identification of A27L dominant epitopes. Antigen-specific IFN-γ ELISPOT in splenocytes from mice (immunized three times, two weeks apart via intramuscular injection) in response to VVWR A27 individual 3-mer overlapped 15-mer overlapping peptides. Each experiment was performed three times and the immune responses among groups of mice are presented as the mean ± standard error of the mean (SEM). A p value of less than 0.05 was considered significant.
    Figure Legend Snippet: A27L-specific cell-mediated immune response against recombinant VVWR determined by ELISPOT analysis. (A) Each group consisted of four female Balb/C mice immunized three times, two weeks apart via intramuscular injection as follows: Naïve (negative control), pVax1 (empty vector backbone control), pA27L (VVWR envelope protein), pA27L+I (VVWR envelope protein + imiquimod), pA27L + R (VVWR envelope protein + resiquimod), pA27LOPT (optimized sequence of the A27L gene) and pA27LOPT+I (the optimized sequence of A27L + imiquimod). (B) Identification of A27L dominant epitopes. Antigen-specific IFN-γ ELISPOT in splenocytes from mice (immunized three times, two weeks apart via intramuscular injection) in response to VVWR A27 individual 3-mer overlapped 15-mer overlapping peptides. Each experiment was performed three times and the immune responses among groups of mice are presented as the mean ± standard error of the mean (SEM). A p value of less than 0.05 was considered significant.

    Techniques Used: Recombinant, Enzyme-linked Immunospot, Mouse Assay, Injection, Negative Control, Plasmid Preparation, Sequencing

    A27L-specific cytokine profile against recombinant VVWR A27L by ELISA. Each group consisted of four female BALB/c mice immunized three times, two weeks apart via intramuscular injection with pA27L, pA27L+I, pA27L+R, pA27LOPT, pA27LOPT+I. Naïve and pVax1 were used as controls. A week after the last immunization, splenocytes from each group of mice were stimulated overnight with VVWR A27-overlapping peptides, and the cytokine-containing supernatants were examined by ELISA. The plate was analyzed by scanning the absorption at 450 nm. The immune responses among groups of mice are presented as the mean ± standard error of the mean (SEM) of at least three independent experiments. A p value of less than 0.05 was considered significant.
    Figure Legend Snippet: A27L-specific cytokine profile against recombinant VVWR A27L by ELISA. Each group consisted of four female BALB/c mice immunized three times, two weeks apart via intramuscular injection with pA27L, pA27L+I, pA27L+R, pA27LOPT, pA27LOPT+I. Naïve and pVax1 were used as controls. A week after the last immunization, splenocytes from each group of mice were stimulated overnight with VVWR A27-overlapping peptides, and the cytokine-containing supernatants were examined by ELISA. The plate was analyzed by scanning the absorption at 450 nm. The immune responses among groups of mice are presented as the mean ± standard error of the mean (SEM) of at least three independent experiments. A p value of less than 0.05 was considered significant.

    Techniques Used: Recombinant, Enzyme-linked Immunosorbent Assay, Mouse Assay, Injection

    Enzymatic digestion of the pA27L plasmid. The A27L and A2L OPT gene was enzymatically digested with HindIII and NotI. Lane 1, 1kb DNA ladder; Lane 2, 100bp DNA ladder; Lane 3, pVax1 uncut; Lane 4 pVax1 cut; Lane 5, pA27L uncut; Lane 6, pA27L cut; Lane 7, pA27LOPT uncut; Lane 8, pA27LOPT cut. The expected band of 434bp corresponding to the A27L gene is highlighted with an arrow.
    Figure Legend Snippet: Enzymatic digestion of the pA27L plasmid. The A27L and A2L OPT gene was enzymatically digested with HindIII and NotI. Lane 1, 1kb DNA ladder; Lane 2, 100bp DNA ladder; Lane 3, pVax1 uncut; Lane 4 pVax1 cut; Lane 5, pA27L uncut; Lane 6, pA27L cut; Lane 7, pA27LOPT uncut; Lane 8, pA27LOPT cut. The expected band of 434bp corresponding to the A27L gene is highlighted with an arrow.

    Techniques Used: Plasmid Preparation

    Humoral-mediated immune response against recombinant VVWR A27L by ELISA. A. Total IgG response after DNA immunization of four mice three times, two weeks apart with A27L, A27L+I, pA27L+R, pA27LOPT, pA27LOPT+I. Naïve and pVax1 were used as controls, One week after the last immunization, sera from each group of mice were diluted at 1:400, and incubated in a 96-well plate previously coated with recombinant VVWR A27 protein. B. Antigen-specific production of each IgG subtype shown was detected from each group. The plate was analyzed by scanning the absorption at 450 nm and the immune responses among groups of mice are presented as the mean ± standard error of the mean (SEM) of at least three independent experiment. A p value of less than 0.05 was considered significant.
    Figure Legend Snippet: Humoral-mediated immune response against recombinant VVWR A27L by ELISA. A. Total IgG response after DNA immunization of four mice three times, two weeks apart with A27L, A27L+I, pA27L+R, pA27LOPT, pA27LOPT+I. Naïve and pVax1 were used as controls, One week after the last immunization, sera from each group of mice were diluted at 1:400, and incubated in a 96-well plate previously coated with recombinant VVWR A27 protein. B. Antigen-specific production of each IgG subtype shown was detected from each group. The plate was analyzed by scanning the absorption at 450 nm and the immune responses among groups of mice are presented as the mean ± standard error of the mean (SEM) of at least three independent experiment. A p value of less than 0.05 was considered significant.

    Techniques Used: Recombinant, Enzyme-linked Immunosorbent Assay, Mouse Assay, Incubation

    9) Product Images from "Polyphenol oxidases in Physcomitrella: functional PPO1 knockout modulates cytokinin-dependent developmentin the moss Physcomitrella patens"

    Article Title: Polyphenol oxidases in Physcomitrella: functional PPO1 knockout modulates cytokinin-dependent developmentin the moss Physcomitrella patens

    Journal: Journal of Experimental Botany

    doi: 10.1093/jxb/ers169

    Recombinant PPO proteins and enzyme activity. (A) Western blot analysis of insoluble (left panel), and soluble protein extracts (right panel) enriched and purified on a Ni-column. Protein extracts of 6h IPTG-induced E. coli cultures expressing His-tagged proteins were separated on a 12.5% SDS gel and detected by an anti-His-tag antibody: lacZ:his (120kDa), TpPPO1:his (59kDa, migrates at 65kDa according to Sullivan et al. , 2004 ), PPO6:his (62kDa). (B) Specific PPO activity of enriched and purified soluble protein extracts determined polarographically using 4-methylcatechol as a substrate. Bar, standard deviation ( n = 3).
    Figure Legend Snippet: Recombinant PPO proteins and enzyme activity. (A) Western blot analysis of insoluble (left panel), and soluble protein extracts (right panel) enriched and purified on a Ni-column. Protein extracts of 6h IPTG-induced E. coli cultures expressing His-tagged proteins were separated on a 12.5% SDS gel and detected by an anti-His-tag antibody: lacZ:his (120kDa), TpPPO1:his (59kDa, migrates at 65kDa according to Sullivan et al. , 2004 ), PPO6:his (62kDa). (B) Specific PPO activity of enriched and purified soluble protein extracts determined polarographically using 4-methylcatechol as a substrate. Bar, standard deviation ( n = 3).

    Techniques Used: Recombinant, Activity Assay, Western Blot, Purification, Expressing, SDS-Gel, Standard Deviation

    10) Product Images from "Streamlined ex vivo and in vivo genome editing in mouse embryos using recombinant adeno-associated viruses"

    Article Title: Streamlined ex vivo and in vivo genome editing in mouse embryos using recombinant adeno-associated viruses

    Journal: Nature Communications

    doi: 10.1038/s41467-017-02706-7

    Recombinant AAV vectors can mediate homology-directed repair (HDR). a Schematic representation of the Tyr locus and location of sgRNA in exon 1. The orange and red lines mark the initiation and termination codons respectively. The green line indicates the location of the sgRNA used to target Tyr . b Strategy to introduce a premature stop codon in the Tyr locus using HDR. The 5′ and 3′ homology arms are marked by a thick line. A G to T nucleotide transversion in the PAM sequence converts a glycine codon (GGA) into a stop codon (TGA) disrupting translation of Tyr . Arrows indicate binding sites of the primers used in PCR-TOPO sequencing. c Strategy to insert the blue fluorescent protein (BFP) gene into the Tyr locus using HDR. Brown and purple arrows depict the binding sites of PCR primers used to confirm the insertion of BFP into Tyr locus. P2A, Porcine teschovirus-1 2A peptide; TAA, Stop codon. d Histogram showing the frequency of single-nucleotide transversion and BFP insertion by HDR using two different mixtures of rAAV vectors. e Analysis of single-nucleotide transversion in individual embryos or pups using PCR-TOPO sequencing. Each bar represents an individual sample. For pups, only DNA from tail snips and ear punches was analyzed. f Confirmation of BFP insertion using PCR. Four out of seven E3.5 embryos tested showed correct insertion of BFP into the Tyr locus. The top panel shows amplification of the 5′-junction of the targeted Tyr locus using a forward primer that binds to genomic DNA upstream of the homology region and a reverse primer that binds to the BFP gene as shown in ( c ). The bottom panel shows amplification of the 3′-junction of the Tyr- edited allele using a forward primer that binds to the BFP gene and a reverse primer that binds to genomic DNA downstream of the homology region
    Figure Legend Snippet: Recombinant AAV vectors can mediate homology-directed repair (HDR). a Schematic representation of the Tyr locus and location of sgRNA in exon 1. The orange and red lines mark the initiation and termination codons respectively. The green line indicates the location of the sgRNA used to target Tyr . b Strategy to introduce a premature stop codon in the Tyr locus using HDR. The 5′ and 3′ homology arms are marked by a thick line. A G to T nucleotide transversion in the PAM sequence converts a glycine codon (GGA) into a stop codon (TGA) disrupting translation of Tyr . Arrows indicate binding sites of the primers used in PCR-TOPO sequencing. c Strategy to insert the blue fluorescent protein (BFP) gene into the Tyr locus using HDR. Brown and purple arrows depict the binding sites of PCR primers used to confirm the insertion of BFP into Tyr locus. P2A, Porcine teschovirus-1 2A peptide; TAA, Stop codon. d Histogram showing the frequency of single-nucleotide transversion and BFP insertion by HDR using two different mixtures of rAAV vectors. e Analysis of single-nucleotide transversion in individual embryos or pups using PCR-TOPO sequencing. Each bar represents an individual sample. For pups, only DNA from tail snips and ear punches was analyzed. f Confirmation of BFP insertion using PCR. Four out of seven E3.5 embryos tested showed correct insertion of BFP into the Tyr locus. The top panel shows amplification of the 5′-junction of the targeted Tyr locus using a forward primer that binds to genomic DNA upstream of the homology region and a reverse primer that binds to the BFP gene as shown in ( c ). The bottom panel shows amplification of the 3′-junction of the Tyr- edited allele using a forward primer that binds to the BFP gene and a reverse primer that binds to genomic DNA downstream of the homology region

    Techniques Used: Recombinant, Introduce, Sequencing, Binding Assay, Polymerase Chain Reaction, Amplification

    11) Product Images from "Elimination of 01/A′–A0 pre-rRNA processing by-product in human cells involves cooperative action of two nuclear exosome-associated nucleases: RRP6 and DIS3"

    Article Title: Elimination of 01/A′–A0 pre-rRNA processing by-product in human cells involves cooperative action of two nuclear exosome-associated nucleases: RRP6 and DIS3

    Journal: RNA

    doi: 10.1261/rna.066589.118

    Expression of catalytically compromised DIS3 or RRP6 variants does not significantly affect rRNA synthesis and ribosome biogenesis in human cells. ( A ) General principle of the utilized cellular model. Plasmids compatible with Flp-In T-REx system from Invitrogen, containing wild-type or mutated variants of FLAG-tagged DIS3 or RRP6 and an EGFP-sh-miRNA fusion (both under the control of a bidirectional tetracycline-regulated promoter) were integrated into the Hek293 Flp-In T-REx cell line genome. The FLAG-tagged DIS3/RRP6 ORF was recoded in a way rendering it insusceptible to sh-miRNA silencing. Upon induction with tetracycline, stable cell lines produced either wild-type or mutated FLAG-tagged protein fusions and sh-miRNA silencing expression of only the respective endogenous gene. ( B ) sh-miRNA efficiently down-regulate expression of endogenous DIS3 and RRP6 at the mRNA level. Quantitative RT-PCR analysis was performed on total RNA isolated from Hek293 Flp-In T-REx cells (Ø) and established model cell lines subjected to induction with tetracycline and producing either DIS3 (WT, RNB MUT, PIN MUT, or DM) or RRP6 (WT or mut) exogenous variants simultaneously with sh-miRNAs targeting respective endogenous transcript. The graph shows results of quantification of three independent experiments. GAPDH mRNA was used for normalization. The expression level is relative to the parental Hek293 Flp-In T-REx cell line. ( C ) Expression of exogenous DIS3 protein variants is higher than endogenous DIS3, whereas levels of exo- and endogenous RRP6 are comparable. Model cell lines (as in panel B ), or parental Hek293 Flp-In T-REx cells (control), were treated with tetracycline. Proteins extracted from cells were separated in SDS-PAGE and transferred onto nitrocellulose membranes, which were stained with Ponceau S-Red and probed with antibodies specific to FLAG epitope, EGFP, DIS3, RRP6, and β-actin (loading control). ( D ) Exogenous DIS3 is overexpressed around five- and 10-fold compared to endogenous protein. Western blot was performed as in panel C , but using various dilutions of the protein sample from cell line-producing DIS3 WT variant. ( E ) Analysis of nascent rRNA synthesis. Model cell lines cultured in a medium containing tetracycline were pulse labeled with 32 P orthophosphoric acid, followed by chase in normal media for varying times (indicated above each lane). RNA was then isolated from the cells, separated in a denaturing agarose-formaldehyde gel and transferred onto nylon membrane. The blot was first stained with methylene blue ( bottom part) and then subjected to phosphorimaging ( upper part). Positions of 28S and 18S rRNA and other visible rRNA species are indicated on the right . In addition, the membrane was probed with hITS2a oligonucleotide ( middle part) to monitor accumulation of 7S pre-rRNA, a known phenotype of DIS3 enzymatic dysfunction. Results of hybridizations with probes h5.8S and h5S ( middle part) and staining of the membrane with methylene blue ( bottom part) are shown to assess sample loading. ( F ) Selected samples from panel E were resolved in denaturing polyacrylamide gel and subsequently subjected to phosphorimaging to visualize synthesis of small RNAs (5.8S, 5S, and tRNAs) at higher resolution. ( G ) Ribosome/polysome profile analysis. Native cytoplasmic extracts were prepared from model cell lines, grown in the presence of tetracycline, following translation inhibition with cycloheximide, and separated by centrifugation in linear sucrose gradients. Graphs show distribution of absorbance at 254 nm from the top ( left ) to the bottom ( right ). Peaks corresponding to individual subunits (40S and 60S), monosomes (80S), and polysomes are indicated.
    Figure Legend Snippet: Expression of catalytically compromised DIS3 or RRP6 variants does not significantly affect rRNA synthesis and ribosome biogenesis in human cells. ( A ) General principle of the utilized cellular model. Plasmids compatible with Flp-In T-REx system from Invitrogen, containing wild-type or mutated variants of FLAG-tagged DIS3 or RRP6 and an EGFP-sh-miRNA fusion (both under the control of a bidirectional tetracycline-regulated promoter) were integrated into the Hek293 Flp-In T-REx cell line genome. The FLAG-tagged DIS3/RRP6 ORF was recoded in a way rendering it insusceptible to sh-miRNA silencing. Upon induction with tetracycline, stable cell lines produced either wild-type or mutated FLAG-tagged protein fusions and sh-miRNA silencing expression of only the respective endogenous gene. ( B ) sh-miRNA efficiently down-regulate expression of endogenous DIS3 and RRP6 at the mRNA level. Quantitative RT-PCR analysis was performed on total RNA isolated from Hek293 Flp-In T-REx cells (Ø) and established model cell lines subjected to induction with tetracycline and producing either DIS3 (WT, RNB MUT, PIN MUT, or DM) or RRP6 (WT or mut) exogenous variants simultaneously with sh-miRNAs targeting respective endogenous transcript. The graph shows results of quantification of three independent experiments. GAPDH mRNA was used for normalization. The expression level is relative to the parental Hek293 Flp-In T-REx cell line. ( C ) Expression of exogenous DIS3 protein variants is higher than endogenous DIS3, whereas levels of exo- and endogenous RRP6 are comparable. Model cell lines (as in panel B ), or parental Hek293 Flp-In T-REx cells (control), were treated with tetracycline. Proteins extracted from cells were separated in SDS-PAGE and transferred onto nitrocellulose membranes, which were stained with Ponceau S-Red and probed with antibodies specific to FLAG epitope, EGFP, DIS3, RRP6, and β-actin (loading control). ( D ) Exogenous DIS3 is overexpressed around five- and 10-fold compared to endogenous protein. Western blot was performed as in panel C , but using various dilutions of the protein sample from cell line-producing DIS3 WT variant. ( E ) Analysis of nascent rRNA synthesis. Model cell lines cultured in a medium containing tetracycline were pulse labeled with 32 P orthophosphoric acid, followed by chase in normal media for varying times (indicated above each lane). RNA was then isolated from the cells, separated in a denaturing agarose-formaldehyde gel and transferred onto nylon membrane. The blot was first stained with methylene blue ( bottom part) and then subjected to phosphorimaging ( upper part). Positions of 28S and 18S rRNA and other visible rRNA species are indicated on the right . In addition, the membrane was probed with hITS2a oligonucleotide ( middle part) to monitor accumulation of 7S pre-rRNA, a known phenotype of DIS3 enzymatic dysfunction. Results of hybridizations with probes h5.8S and h5S ( middle part) and staining of the membrane with methylene blue ( bottom part) are shown to assess sample loading. ( F ) Selected samples from panel E were resolved in denaturing polyacrylamide gel and subsequently subjected to phosphorimaging to visualize synthesis of small RNAs (5.8S, 5S, and tRNAs) at higher resolution. ( G ) Ribosome/polysome profile analysis. Native cytoplasmic extracts were prepared from model cell lines, grown in the presence of tetracycline, following translation inhibition with cycloheximide, and separated by centrifugation in linear sucrose gradients. Graphs show distribution of absorbance at 254 nm from the top ( left ) to the bottom ( right ). Peaks corresponding to individual subunits (40S and 60S), monosomes (80S), and polysomes are indicated.

    Techniques Used: Expressing, Stable Transfection, Produced, Quantitative RT-PCR, Isolation, SDS Page, Staining, FLAG-tag, Western Blot, Variant Assay, Cell Culture, Labeling, Inhibition, Centrifugation

    12) Product Images from "Host PGRP Gene Expression and Bacterial Release in Endosymbiosis of the Weevil Sitophilus zeamais"

    Article Title: Host PGRP Gene Expression and Bacterial Release in Endosymbiosis of the Weevil Sitophilus zeamais

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.00942-06

    Quantification of wPGRP gene transcripts from control and challenged aposymbiotic larvae. (A) Shown are the images of the Northern blot hybridized with wPGRP and with β-actin probes. Cal, calibrator sample; C, unchallenged aposymbiotic larvae; S, mock-infected larvae; E and P are larvae infected with E. coli and P. aeruginosa , respectively; numbers 1, 2, and 3 indicate sample repetitions. Larvae were incubated for 1, 2, 6, and 12 h following the treatment. (B) Levels of wPGRP gene transcripts normalized with the expression of the β-actin gene. Data (expressed in arbitrary units) are means of three independent repetitions normalized with the relative intensity of the β-actin gene. Bars represent the standard deviations. Data were analyzed with ANOVA (see Results).
    Figure Legend Snippet: Quantification of wPGRP gene transcripts from control and challenged aposymbiotic larvae. (A) Shown are the images of the Northern blot hybridized with wPGRP and with β-actin probes. Cal, calibrator sample; C, unchallenged aposymbiotic larvae; S, mock-infected larvae; E and P are larvae infected with E. coli and P. aeruginosa , respectively; numbers 1, 2, and 3 indicate sample repetitions. Larvae were incubated for 1, 2, 6, and 12 h following the treatment. (B) Levels of wPGRP gene transcripts normalized with the expression of the β-actin gene. Data (expressed in arbitrary units) are means of three independent repetitions normalized with the relative intensity of the β-actin gene. Bars represent the standard deviations. Data were analyzed with ANOVA (see Results).

    Techniques Used: Northern Blot, Infection, Incubation, Expressing

    Bacterial growth in aposymbiotic infected larvae. Larvae were infected with either E. coli or P. aeruginosa . The number of bacteria was measured at 2, 6, 12, and 24 h after infection. Data are expressed as CFU (CFU per larva) and represent the means of two samples of five larvae.
    Figure Legend Snippet: Bacterial growth in aposymbiotic infected larvae. Larvae were infected with either E. coli or P. aeruginosa . The number of bacteria was measured at 2, 6, 12, and 24 h after infection. Data are expressed as CFU (CFU per larva) and represent the means of two samples of five larvae.

    Techniques Used: Infection

    13) Product Images from "Improved antifouling properties and selective biofunctionalization of stainless steel by employing heterobifunctional silane-polyethylene glycol overlayers and avidin-biotin technology"

    Article Title: Improved antifouling properties and selective biofunctionalization of stainless steel by employing heterobifunctional silane-polyethylene glycol overlayers and avidin-biotin technology

    Journal: Scientific Reports

    doi: 10.1038/srep29324

    Protein adsorption on SS-SPC. ( a ) Fluorescence microscopy images of Alexa Fluor® 488 labeled avidin and fibronectin adsorbed on unmodified SS (SS-ctrl) and SS-SPC in different conditions. The used protein concentration was either 3 μg/ml (C1) or 30 μg/ml (C2), and the exposure time 1 h (T1) or 3 h (T2). Scale bars 100 μm. ( b,c ) The boxplots show the mean fluorescence intensities of the adsorbed avidin and fibronectin, respectively, on SS-SPC (SPC) and unmodified SS (Ctrl) in the conditions depicted in ( a ).
    Figure Legend Snippet: Protein adsorption on SS-SPC. ( a ) Fluorescence microscopy images of Alexa Fluor® 488 labeled avidin and fibronectin adsorbed on unmodified SS (SS-ctrl) and SS-SPC in different conditions. The used protein concentration was either 3 μg/ml (C1) or 30 μg/ml (C2), and the exposure time 1 h (T1) or 3 h (T2). Scale bars 100 μm. ( b,c ) The boxplots show the mean fluorescence intensities of the adsorbed avidin and fibronectin, respectively, on SS-SPC (SPC) and unmodified SS (Ctrl) in the conditions depicted in ( a ).

    Techniques Used: Adsorption, Fluorescence, Microscopy, Labeling, Avidin-Biotin Assay, Protein Concentration

    14) Product Images from "Ubiquitin E3 ligase FIEL1 regulates fibrotic lung injury through SUMO-E3 ligase PIAS4"

    Article Title: Ubiquitin E3 ligase FIEL1 regulates fibrotic lung injury through SUMO-E3 ligase PIAS4

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20151229

    PIAS4 phosphorylation by PKCζ is required for FIEL1 binding. (A) Endogenous PIAS4 was immunoprecipitated and immunoblotted for Erk1, PKCα, and PKCζ ( n = 2). (B) MLE cells were transfected with increasing amounts of PKCζ or JNK1 plasmids for 18 h before PIAS4 immunoblotting ( n = 2). (C) PIAS4 protein half-life determination with CON shRNA or PKCζ shRNA expression ( n = 3). (D) PIAS4 protein half-life determination with Empty or PKCζ plasmid overexpression ( n = 3). (E and F) MRC5 cells were treated with TGFβ in a time or dose-dependent manner; cells were collected and immunoblotted for FIEL1, PIAS4, PKCζ, and p-PKCζ (Thr410). Endogenous PIAS4 was also immunoprecipitated and followed by PKCζ, PKCα, phosphoserine, and phosphothreonine immunoblotting ( n = 2). (G) In vitro PKCζ kinase assay. Recombinant PKCζ (Enzo) was used as the kinase, and V5-tagged PIAS4 was synthesized via TnT in vitro kits (Promega), purified by HIS pulldown, and used as the substrate. The kinase reactions were incubated at 37°C for 2 h, and products were resolved by SDS-PAGE and processed for autoradiography either by using Personal Molecular Imager (Bio-Rad Laboratories) or immunoblotting for V5 to visualize the substrate input. *, heat inactivated PKCζ ( n = 2). (H) Immunoblots showing levels of FIEL1, PKCζ, p-PKCζ (Thr410), and PIAS4 protein in 293T cells transfected with either CON shRNA or PKCζ shRNA, followed by a TGFβ dose treatment. Endogenous PIAS4 was also immunoprecipitated and followed by PKCζ, phosphoserine, and phosphothreonine immunoblotting. (I) 293T cells were transfected with WT, S14A, S18A, or S14/18A PIAS4 before being treated with a dose course of TGFβ. Cells were then collected and assayed for V5-PIAS4 immunoblotting. Overexpressed V5-PIAS4 was also immunoprecipitated using a V5 antibody and followed by phosphoserine immunoblotting ( n = 2). (J) Four biotin-labeled PIAS4 peptides were bound to streptavidin and served as the bait for FIEL1 binding. After washing, proteins were eluted and immunoblotted for FIEL1-V5 ( n = 2).
    Figure Legend Snippet: PIAS4 phosphorylation by PKCζ is required for FIEL1 binding. (A) Endogenous PIAS4 was immunoprecipitated and immunoblotted for Erk1, PKCα, and PKCζ ( n = 2). (B) MLE cells were transfected with increasing amounts of PKCζ or JNK1 plasmids for 18 h before PIAS4 immunoblotting ( n = 2). (C) PIAS4 protein half-life determination with CON shRNA or PKCζ shRNA expression ( n = 3). (D) PIAS4 protein half-life determination with Empty or PKCζ plasmid overexpression ( n = 3). (E and F) MRC5 cells were treated with TGFβ in a time or dose-dependent manner; cells were collected and immunoblotted for FIEL1, PIAS4, PKCζ, and p-PKCζ (Thr410). Endogenous PIAS4 was also immunoprecipitated and followed by PKCζ, PKCα, phosphoserine, and phosphothreonine immunoblotting ( n = 2). (G) In vitro PKCζ kinase assay. Recombinant PKCζ (Enzo) was used as the kinase, and V5-tagged PIAS4 was synthesized via TnT in vitro kits (Promega), purified by HIS pulldown, and used as the substrate. The kinase reactions were incubated at 37°C for 2 h, and products were resolved by SDS-PAGE and processed for autoradiography either by using Personal Molecular Imager (Bio-Rad Laboratories) or immunoblotting for V5 to visualize the substrate input. *, heat inactivated PKCζ ( n = 2). (H) Immunoblots showing levels of FIEL1, PKCζ, p-PKCζ (Thr410), and PIAS4 protein in 293T cells transfected with either CON shRNA or PKCζ shRNA, followed by a TGFβ dose treatment. Endogenous PIAS4 was also immunoprecipitated and followed by PKCζ, phosphoserine, and phosphothreonine immunoblotting. (I) 293T cells were transfected with WT, S14A, S18A, or S14/18A PIAS4 before being treated with a dose course of TGFβ. Cells were then collected and assayed for V5-PIAS4 immunoblotting. Overexpressed V5-PIAS4 was also immunoprecipitated using a V5 antibody and followed by phosphoserine immunoblotting ( n = 2). (J) Four biotin-labeled PIAS4 peptides were bound to streptavidin and served as the bait for FIEL1 binding. After washing, proteins were eluted and immunoblotted for FIEL1-V5 ( n = 2).

    Techniques Used: Binding Assay, Immunoprecipitation, Transfection, shRNA, Expressing, Plasmid Preparation, Over Expression, In Vitro, Kinase Assay, Recombinant, Synthesized, Purification, Incubation, SDS Page, Autoradiography, Western Blot, Labeling

    15) Product Images from "HybriFree: a robust and rapid method for the development of monoclonal antibodies from different host species"

    Article Title: HybriFree: a robust and rapid method for the development of monoclonal antibodies from different host species

    Journal: BMC Biotechnology

    doi: 10.1186/s12896-016-0232-6

    Construction and screening of intact IgG molecules. a The pQMCF IgG vector was constructed using single-step CPEC joining of 4 fragments: VH, VL, promoters/leaders and vector. The antibody heavy and light chains are expressed from the resulting vector as separate proteins that assemble naturally into IgG molecules secreted from mammalian cells. b . Western blot analysis of rabbit IgG secretion from CHOEBNALT85 cells transfected with pQMCF IgG library pool DNA constructed from VH and VL regions from a rabbit immunized with mouse CD48 protein. Goat polyclonal antibody against rabbit IgG heavy chain was used for the detection of free heavy chain in reduced sample conditions (DTT+) and of the assembled IgG molecule in non-reduced (DTT-) sample. c Mouse CD48 ELISA results obtained using serial dilutions of the same sample of library pool transfection media as primary antibody. d Distribution of positive and negative clones obtained from the screening of the library pool showed in the panel c . Three positive and two negative clones selected for sequencing are indicated. e Alignment of VL domain amino acid sequences of the positive and negative clones. CDR regions are underlined
    Figure Legend Snippet: Construction and screening of intact IgG molecules. a The pQMCF IgG vector was constructed using single-step CPEC joining of 4 fragments: VH, VL, promoters/leaders and vector. The antibody heavy and light chains are expressed from the resulting vector as separate proteins that assemble naturally into IgG molecules secreted from mammalian cells. b . Western blot analysis of rabbit IgG secretion from CHOEBNALT85 cells transfected with pQMCF IgG library pool DNA constructed from VH and VL regions from a rabbit immunized with mouse CD48 protein. Goat polyclonal antibody against rabbit IgG heavy chain was used for the detection of free heavy chain in reduced sample conditions (DTT+) and of the assembled IgG molecule in non-reduced (DTT-) sample. c Mouse CD48 ELISA results obtained using serial dilutions of the same sample of library pool transfection media as primary antibody. d Distribution of positive and negative clones obtained from the screening of the library pool showed in the panel c . Three positive and two negative clones selected for sequencing are indicated. e Alignment of VL domain amino acid sequences of the positive and negative clones. CDR regions are underlined

    Techniques Used: Plasmid Preparation, Construct, Western Blot, Transfection, Enzyme-linked Immunosorbent Assay, Clone Assay, Sequencing

    16) Product Images from "Study of model systems to test the potential function of Artemia group 1 late embryogenesis abundant (LEA) proteins"

    Article Title: Study of model systems to test the potential function of Artemia group 1 late embryogenesis abundant (LEA) proteins

    Journal: Cell Stress & Chaperones

    doi: 10.1007/s12192-015-0647-3

    Effect of sorbitol and IPTG on growth of E. coli Top10F’ transformed with pCR2.1/AfrLEA-1 . Cells were grown overnight in LB medium (with ampicillin) and their concentration was determined. An equal number of cells (equivalent to 0.001 absorbance
    Figure Legend Snippet: Effect of sorbitol and IPTG on growth of E. coli Top10F’ transformed with pCR2.1/AfrLEA-1 . Cells were grown overnight in LB medium (with ampicillin) and their concentration was determined. An equal number of cells (equivalent to 0.001 absorbance

    Techniques Used: Transformation Assay, Concentration Assay

    Growth kinetics of E. coli Top10F’ transformed with an inducible AfrLEA-1 gene in plasmid pCR2.1 (tE1C-3). a Growth kinetics over 9.5 h at 37 °C in the presence or absence of IPTG. The arrow indicates the time of IPTG addition.
    Figure Legend Snippet: Growth kinetics of E. coli Top10F’ transformed with an inducible AfrLEA-1 gene in plasmid pCR2.1 (tE1C-3). a Growth kinetics over 9.5 h at 37 °C in the presence or absence of IPTG. The arrow indicates the time of IPTG addition.

    Techniques Used: Transformation Assay, Plasmid Preparation

    Growth of E. coli transformed with different plasmid constructs. a Growth of E. coli Top10 after 22–23 h incubation at 37 °C in LB medium. pCR2.1 clone with the empty vector, B25 control clone with E1A sequence in reverse
    Figure Legend Snippet: Growth of E. coli transformed with different plasmid constructs. a Growth of E. coli Top10 after 22–23 h incubation at 37 °C in LB medium. pCR2.1 clone with the empty vector, B25 control clone with E1A sequence in reverse

    Techniques Used: Transformation Assay, Plasmid Preparation, Construct, Incubation, Sequencing

    17) Product Images from "Phylogenetically widespread alternative splicing at unusual GYNGYN donors"

    Article Title: Phylogenetically widespread alternative splicing at unusual GYNGYN donors

    Journal: Genome Biology

    doi: 10.1186/gb-2006-7-7-r65

    Alternative splicing at the tandem donor of exon 21 of (a) human STAT3 and (b) mouse Stat3 . Electropherograms are shown for direct sequencing of RT-PCR amplicons (e+i) and sequencing of isolated clones representing e and i transcripts (e and i, respectively). The cursor is positioned on the nucleotide upstream of the conserved GTAGTT motif. Numbers and ratios of clones representing e and i transcripts are given for human and mouse kidney (e:i).
    Figure Legend Snippet: Alternative splicing at the tandem donor of exon 21 of (a) human STAT3 and (b) mouse Stat3 . Electropherograms are shown for direct sequencing of RT-PCR amplicons (e+i) and sequencing of isolated clones representing e and i transcripts (e and i, respectively). The cursor is positioned on the nucleotide upstream of the conserved GTAGTT motif. Numbers and ratios of clones representing e and i transcripts are given for human and mouse kidney (e:i).

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

    18) Product Images from "Genome-Wide Transposon Mutagenesis of Borrelia burgdorferi for Identification of Phenotypic Mutants"

    Article Title: Genome-Wide Transposon Mutagenesis of Borrelia burgdorferi for Identification of Phenotypic Mutants

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.70.10.5973-5979.2004

    Transposon mutagenesis system for B. burgdorferi . Suicide vector pMarGent was electroporated into competent B. burgdorferi cells, allowing transient expression of the Himar1 transposase (1). After transposition, the Himar1 gene remains on a DNA fragment that is presumably degraded by intracellular nucleases (2). Mutants were selected in the presence of gentamicin, and those with desired phenotypes were transferred to liquid culture for DNA isolation. The B. burgdorferi DNA flanking the transposon insertion site was recovered by digestion with HindIII, an enzyme that does not cut within the transposon (3 and 4); self-ligation; and transformation into E. coli (5). Purified plasmid DNA was then isolated from E. coli clones and sequenced. IR, inverted repeat.
    Figure Legend Snippet: Transposon mutagenesis system for B. burgdorferi . Suicide vector pMarGent was electroporated into competent B. burgdorferi cells, allowing transient expression of the Himar1 transposase (1). After transposition, the Himar1 gene remains on a DNA fragment that is presumably degraded by intracellular nucleases (2). Mutants were selected in the presence of gentamicin, and those with desired phenotypes were transferred to liquid culture for DNA isolation. The B. burgdorferi DNA flanking the transposon insertion site was recovered by digestion with HindIII, an enzyme that does not cut within the transposon (3 and 4); self-ligation; and transformation into E. coli (5). Purified plasmid DNA was then isolated from E. coli clones and sequenced. IR, inverted repeat.

    Techniques Used: Mutagenesis, Plasmid Preparation, Expressing, DNA Extraction, Ligation, Transformation Assay, Purification, Isolation, Clone Assay

    19) Product Images from "Functional Analysis of NtZIP4B and Zn Status-Dependent Expression Pattern of Tobacco ZIP Genes"

    Article Title: Functional Analysis of NtZIP4B and Zn Status-Dependent Expression Pattern of Tobacco ZIP Genes

    Journal: Frontiers in Plant Science

    doi: 10.3389/fpls.2018.01984

    Complementation by NtZIP4B cDNA of yeast mutants defective in metal uptake on selective media. Yeast cells: DY1457, Δzrt1zrt2 (defective in Zn uptake) were transformed with empty vectors pAG426 as a control, or with vectors carrying tobacco gene NtZIP4B with the stop codon pAG426-ZIP4B-STOP (pAG426-ZIP4B). Yeast cultures were adjusted to an OD600 of 0.2 and 5 μl of serial dilutions (from left to right in each panel) was spotted on SC-URA medium containing 2% (w/v) galactose solidified with 2% agar, supplemented with EGTA (A) , CdCl 2 (B) or 0.2 mM Zn (control). The plates were incubated for 3–6 days at 30°C. The images are representative of three independent experiments taken after 5 days of growth (A) and 3 days of growth (B) .
    Figure Legend Snippet: Complementation by NtZIP4B cDNA of yeast mutants defective in metal uptake on selective media. Yeast cells: DY1457, Δzrt1zrt2 (defective in Zn uptake) were transformed with empty vectors pAG426 as a control, or with vectors carrying tobacco gene NtZIP4B with the stop codon pAG426-ZIP4B-STOP (pAG426-ZIP4B). Yeast cultures were adjusted to an OD600 of 0.2 and 5 μl of serial dilutions (from left to right in each panel) was spotted on SC-URA medium containing 2% (w/v) galactose solidified with 2% agar, supplemented with EGTA (A) , CdCl 2 (B) or 0.2 mM Zn (control). The plates were incubated for 3–6 days at 30°C. The images are representative of three independent experiments taken after 5 days of growth (A) and 3 days of growth (B) .

    Techniques Used: Transformation Assay, Incubation

    20) Product Images from "Identification of a nicotinamide/nicotinate mononucleotide adenylyltransferase in Giardia lamblia (GlNMNAT)"

    Article Title: Identification of a nicotinamide/nicotinate mononucleotide adenylyltransferase in Giardia lamblia (GlNMNAT)

    Journal: Biochimie Open

    doi: 10.1016/j.biopen.2015.11.001

    GlNMNAT enzyme preparation for kinetic experiments . Purity estimated by means of densitometry using the Image J software. Enzyme enrichment calculated taking in account the activity of the soluble fraction and the eluate. Activity in pmol ∗ s −1 ∗ µg −1 or pkat ∗ µg −1 .
    Figure Legend Snippet: GlNMNAT enzyme preparation for kinetic experiments . Purity estimated by means of densitometry using the Image J software. Enzyme enrichment calculated taking in account the activity of the soluble fraction and the eluate. Activity in pmol ∗ s −1 ∗ µg −1 or pkat ∗ µg −1 .

    Techniques Used: Software, Activity Assay

    Amplification of the NMNAT coding region of Giardia lamblia . (1) Ladder 100 bp (fermentas). (2) Negative control (without DNA template). (3) Amplification of glnmnat from genomic DNA. Agarose gel in 1.5% TBE, ethidium bromide staining at 0.05%.
    Figure Legend Snippet: Amplification of the NMNAT coding region of Giardia lamblia . (1) Ladder 100 bp (fermentas). (2) Negative control (without DNA template). (3) Amplification of glnmnat from genomic DNA. Agarose gel in 1.5% TBE, ethidium bromide staining at 0.05%.

    Techniques Used: Amplification, Negative Control, Agarose Gel Electrophoresis, Staining

    Results of a direct enzyme assay using nicotinamide mononucleotide (NMN) or nicotinic acid mononucleotide (NAMN) as a substrate. NMN: (A) Positive control (B) Negative control (C) Reaction mixture with His-GlNMNAT. NAMN: (D) Positive control (E) Negative control (F) Reaction mixture with His-GlNMNAT.
    Figure Legend Snippet: Results of a direct enzyme assay using nicotinamide mononucleotide (NMN) or nicotinic acid mononucleotide (NAMN) as a substrate. NMN: (A) Positive control (B) Negative control (C) Reaction mixture with His-GlNMNAT. NAMN: (D) Positive control (E) Negative control (F) Reaction mixture with His-GlNMNAT.

    Techniques Used: Enzymatic Assay, Positive Control, Negative Control

    Expression of recombinant His-GlNMNAT . (A) SDS-PAGE, Coomassie blue staining. (B) Immunoblotting on nitrocellulose membrane. (1) Molecular weight marker (MWM). (2) Uninduced BL21 cells. (3) Total cells induced. (4) Insoluble fraction. (5) Soluble fraction.
    Figure Legend Snippet: Expression of recombinant His-GlNMNAT . (A) SDS-PAGE, Coomassie blue staining. (B) Immunoblotting on nitrocellulose membrane. (1) Molecular weight marker (MWM). (2) Uninduced BL21 cells. (3) Total cells induced. (4) Insoluble fraction. (5) Soluble fraction.

    Techniques Used: Expressing, Recombinant, SDS Page, Staining, Molecular Weight, Marker

    Enrichment of His-GlNMNAT recombinant protein from the soluble fraction by nickel affinity . (A) SDS-PAGE, Coomassie blue staining. (B) Immunoblotting on nitrocellulose membrane. (1) Molecular weight marker (MWM). (2) Soluble fraction. (3) Unbound proteins. (4) Wash 15. (5,6) Eluates 50 mM Imidazole. (7,8) Eluates 80 mM Imidazole. (9,10) Eluates 250 mM Imidazole.
    Figure Legend Snippet: Enrichment of His-GlNMNAT recombinant protein from the soluble fraction by nickel affinity . (A) SDS-PAGE, Coomassie blue staining. (B) Immunoblotting on nitrocellulose membrane. (1) Molecular weight marker (MWM). (2) Soluble fraction. (3) Unbound proteins. (4) Wash 15. (5,6) Eluates 50 mM Imidazole. (7,8) Eluates 80 mM Imidazole. (9,10) Eluates 250 mM Imidazole.

    Techniques Used: Recombinant, SDS Page, Staining, Molecular Weight, Marker

    Multiple sequence alignment of 16 homologous NMNAT proteins from phylogenetically divergent organisms with GlNMNAT isoenzymes . The percentage of conservation is displayed throughout the sequence in bars. Alignment was done with the ClustalO algorithm in the CLC Sequence Viewer v7.0.2 program (CLCBio A/S, Additional Alignments plugin v.1.5.1).
    Figure Legend Snippet: Multiple sequence alignment of 16 homologous NMNAT proteins from phylogenetically divergent organisms with GlNMNAT isoenzymes . The percentage of conservation is displayed throughout the sequence in bars. Alignment was done with the ClustalO algorithm in the CLC Sequence Viewer v7.0.2 program (CLCBio A/S, Additional Alignments plugin v.1.5.1).

    Techniques Used: Sequencing

    GlNMNAT tertiary structure model . (A) Three-dimensional GlNMNAT model (I-TASSER [15] , [16] ). A Rossmann-type fold, characteristic of nucleotide-binding proteins, is observed. C-score: −5
    Figure Legend Snippet: GlNMNAT tertiary structure model . (A) Three-dimensional GlNMNAT model (I-TASSER [15] , [16] ). A Rossmann-type fold, characteristic of nucleotide-binding proteins, is observed. C-score: −5

    Techniques Used: Binding Assay

    21) Product Images from "Geno- and phenotypic characteristics of a transfected Babesia bovis 6-Cys-E knockout clonal line"

    Article Title: Geno- and phenotypic characteristics of a transfected Babesia bovis 6-Cys-E knockout clonal line

    Journal: Parasites & Vectors

    doi: 10.1186/s13071-017-2143-3

    Comparative in vitro culture growth curves of B. bovis T3Bo, transfected 6-Cys EKO, and 6-Cys EKO- cln parasite lines with and without addition of inhibitory doses of blasticidin. The data are expressed as arithmetic means ± standard deviation (SD). P
    Figure Legend Snippet: Comparative in vitro culture growth curves of B. bovis T3Bo, transfected 6-Cys EKO, and 6-Cys EKO- cln parasite lines with and without addition of inhibitory doses of blasticidin. The data are expressed as arithmetic means ± standard deviation (SD). P

    Techniques Used: In Vitro, Transfection, Standard Deviation

    In vitro neutralisation assay performed on Mo7 strain ( a ), T3Bo strain ( b ), EKO- cln ( c ), and EKO ( d ). Normalised PPE values (Y axis) obtained from B. bovis culture at 72 h in the presence or absence of different sera as indicated on the X-axis. Error bars indicate standard deviations for each sample tested from triplicate culture. Data from each B. bovis strain tested, and data from each treatment (pre-immune Bbo 6-Cys E and rabbit anti-Bbo 6-Cys- E ) were compared using ANOVA analysis. (*) represent P
    Figure Legend Snippet: In vitro neutralisation assay performed on Mo7 strain ( a ), T3Bo strain ( b ), EKO- cln ( c ), and EKO ( d ). Normalised PPE values (Y axis) obtained from B. bovis culture at 72 h in the presence or absence of different sera as indicated on the X-axis. Error bars indicate standard deviations for each sample tested from triplicate culture. Data from each B. bovis strain tested, and data from each treatment (pre-immune Bbo 6-Cys E and rabbit anti-Bbo 6-Cys- E ) were compared using ANOVA analysis. (*) represent P

    Techniques Used: In Vitro

    Schematic representation of ( a ) the structure of the 6-Cys E gene locus, ( b ) transfection plasmid p6-Cys-EKO (GenBank KX247384) and, ( c ) 6 Cys E knock out gene
    Figure Legend Snippet: Schematic representation of ( a ) the structure of the 6-Cys E gene locus, ( b ) transfection plasmid p6-Cys-EKO (GenBank KX247384) and, ( c ) 6 Cys E knock out gene

    Techniques Used: Transfection, Plasmid Preparation, Knock-Out

    Detection of the expression of egfp in B. bovis 6-Cys EKO mutant line by fluorescence microscopy. Upper panels: ( left to right ) represents transfected parasites of the EKO Cln line stained with DAPI, fluorescent light, brightfield and a merged image respectively. Lower panel identical images obtained using the control non-transfected parental parasite line T3Bo. Scale-bars : 5 μm
    Figure Legend Snippet: Detection of the expression of egfp in B. bovis 6-Cys EKO mutant line by fluorescence microscopy. Upper panels: ( left to right ) represents transfected parasites of the EKO Cln line stained with DAPI, fluorescent light, brightfield and a merged image respectively. Lower panel identical images obtained using the control non-transfected parental parasite line T3Bo. Scale-bars : 5 μm

    Techniques Used: Expressing, Mutagenesis, Fluorescence, Microscopy, Transfection, Staining

    Southern blot analysis of gDNA extracted from non-transfected T3Bo,6-Cys EKO, and 6-CysEKO- cln , using Dig-labeled probes against 6-Cys E gene 3′ end probe ( a ), egfp-bsd probe ( b ), Ef-α probe ( c ) and ampicillin probe ( d ). Each sample was analysed using undigested ( - ), or BglII digested (+) gDNA. M: molecular marker
    Figure Legend Snippet: Southern blot analysis of gDNA extracted from non-transfected T3Bo,6-Cys EKO, and 6-CysEKO- cln , using Dig-labeled probes against 6-Cys E gene 3′ end probe ( a ), egfp-bsd probe ( b ), Ef-α probe ( c ) and ampicillin probe ( d ). Each sample was analysed using undigested ( - ), or BglII digested (+) gDNA. M: molecular marker

    Techniques Used: Southern Blot, Transfection, Labeling, Marker

    PCR analysis for integration of egfp-bsd in transfected B. bovis . a Top panel: schematic representation of the intact 6-Cys E locus in the T3Bo strain and the 6-Cys EKO- cln parasites as deduced from full genome sequence of 6-Cys EKO- cln parasites. The red coloured lines represent gDNA sequences that are not a part of the targeted region included in the transfection plasmid p6-Cys-EKO . Lower panel: agarose electrophoresis analysis performed on the PCR products corresponding to each of the fragments numbers 1–7 in the upper panel. b Comparative PCR analysis performed on the line EKO- cln , T3Bo, and EKO, using RAP-1, 6-Cys E , egfpbsd , and IGE - Pro primer sets. SM represents standard molecular weight 10 Kbp
    Figure Legend Snippet: PCR analysis for integration of egfp-bsd in transfected B. bovis . a Top panel: schematic representation of the intact 6-Cys E locus in the T3Bo strain and the 6-Cys EKO- cln parasites as deduced from full genome sequence of 6-Cys EKO- cln parasites. The red coloured lines represent gDNA sequences that are not a part of the targeted region included in the transfection plasmid p6-Cys-EKO . Lower panel: agarose electrophoresis analysis performed on the PCR products corresponding to each of the fragments numbers 1–7 in the upper panel. b Comparative PCR analysis performed on the line EKO- cln , T3Bo, and EKO, using RAP-1, 6-Cys E , egfpbsd , and IGE - Pro primer sets. SM represents standard molecular weight 10 Kbp

    Techniques Used: Polymerase Chain Reaction, Transfection, Sequencing, Plasmid Preparation, Electrophoresis, Molecular Weight

    22) Product Images from "Overproduction, purification, and characterization of nanosized polyphosphate bodies from Synechococcus sp. PCC 7002"

    Article Title: Overproduction, purification, and characterization of nanosized polyphosphate bodies from Synechococcus sp. PCC 7002

    Journal: Microbial Cell Factories

    doi: 10.1186/s12934-018-0870-6

    Electrophoresis results of the construction of transgenic strains on a 1% agarose gel. A PCR products of target gene; B purified target gene from PCR products; C lane a: ppk +pEASY-Blunt vector; line b: ppk +pEASY-Blunt vector digested with Hin dIII and Kpn I restriction enzymes; line c: pSyn_1 vector; line d: pSyn_1 vector digested with Hin dIII and Kpn I restriction enzymes; D lane a: ppk +pSyn_1 vector; line b: ppk +pSyn_1 vector digested with Hin dIII or Kpn I restriction enzymes; line c: ppk +pSyn_1 vector digested with Hin dIII and Kpn I restriction enzymes; E PCR products from ppk -type strain plasmid (lane a and b); M in A , B , E was 5 Kb DNA Marker, in C , D was 1 Kb DNA Ladder
    Figure Legend Snippet: Electrophoresis results of the construction of transgenic strains on a 1% agarose gel. A PCR products of target gene; B purified target gene from PCR products; C lane a: ppk +pEASY-Blunt vector; line b: ppk +pEASY-Blunt vector digested with Hin dIII and Kpn I restriction enzymes; line c: pSyn_1 vector; line d: pSyn_1 vector digested with Hin dIII and Kpn I restriction enzymes; D lane a: ppk +pSyn_1 vector; line b: ppk +pSyn_1 vector digested with Hin dIII or Kpn I restriction enzymes; line c: ppk +pSyn_1 vector digested with Hin dIII and Kpn I restriction enzymes; E PCR products from ppk -type strain plasmid (lane a and b); M in A , B , E was 5 Kb DNA Marker, in C , D was 1 Kb DNA Ladder

    Techniques Used: Electrophoresis, Transgenic Assay, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Purification, Plasmid Preparation, Marker

    23) Product Images from "Candidate Antigens for Q Fever Serodiagnosis Revealed by Immunoscreening of a Coxiella burnetii Protein Microarray ▿"

    Article Title: Candidate Antigens for Q Fever Serodiagnosis Revealed by Immunoscreening of a Coxiella burnetii Protein Microarray ▿

    Journal:

    doi: 10.1128/CVI.00300-08

    Protein microarray analysis of the human humoral response to C. burnetii infection or vaccination. Microarrays were probed with paired “early” and “late” acute Q fever patient sera that was obtained at 20 and 96 days, respectively,
    Figure Legend Snippet: Protein microarray analysis of the human humoral response to C. burnetii infection or vaccination. Microarrays were probed with paired “early” and “late” acute Q fever patient sera that was obtained at 20 and 96 days, respectively,

    Techniques Used: Microarray, Infection

    Schematic of TAP product synthesis. C. burnetii Nine Mile ORFs were amplified in a first round of PCR using genomic DNA as a template and PCR primers consisting of gene-specific sequence and common 5′ overlap regions. TAP products were generated
    Figure Legend Snippet: Schematic of TAP product synthesis. C. burnetii Nine Mile ORFs were amplified in a first round of PCR using genomic DNA as a template and PCR primers consisting of gene-specific sequence and common 5′ overlap regions. TAP products were generated

    Techniques Used: Amplification, Polymerase Chain Reaction, Sequencing, Generated

    24) Product Images from "Distribution of Florfenicol Resistance Genes fexA and cfr among Chloramphenicol-Resistant Staphylococcus Isolates"

    Article Title: Distribution of Florfenicol Resistance Genes fexA and cfr among Chloramphenicol-Resistant Staphylococcus Isolates

    Journal:

    doi: 10.1128/AAC.50.4.1156-1163.2006

    (a) PCR amplicons specific for the detection of transposase genes tnpA (lane 1), tnpB (lane 2), and tnpC (lane 3), the linkage between tnpB-fexA (lane 4), an internal 5,741-bp fragment representing the almost complete Tn 558 (lane 5), and a circular intermediate
    Figure Legend Snippet: (a) PCR amplicons specific for the detection of transposase genes tnpA (lane 1), tnpB (lane 2), and tnpC (lane 3), the linkage between tnpB-fexA (lane 4), an internal 5,741-bp fragment representing the almost complete Tn 558 (lane 5), and a circular intermediate

    Techniques Used: Polymerase Chain Reaction

    25) Product Images from "UBC9 Autosumoylation Negatively Regulates Sumoylation of Septins in Saccharomyces cerevisiae *"

    Article Title: UBC9 Autosumoylation Negatively Regulates Sumoylation of Septins in Saccharomyces cerevisiae *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.234914

    Modification of yeast Ubc9 by Smt3. A , yeast Ubc9 autosumoylation in vitro. In vitro sumoylation assay was performed in the presence of 400 ng of Aos1-Uba2, 1 μg of Ubc9, 1 μg of Smt3, and 5 m m ATP at 37 °C for 0 or 30 min. The
    Figure Legend Snippet: Modification of yeast Ubc9 by Smt3. A , yeast Ubc9 autosumoylation in vitro. In vitro sumoylation assay was performed in the presence of 400 ng of Aos1-Uba2, 1 μg of Ubc9, 1 μg of Smt3, and 5 m m ATP at 37 °C for 0 or 30 min. The

    Techniques Used: Modification, In Vitro

    Ubc9 autosumoylation negatively regulates septin sumoylation of septins in vivo . Cdc3 sumoylation in vivo is shown. Cdc3 sumoylation was analyzed in cells grown to log phase ( lanes 1–4 ) or arrested in S or G 2 /M phase with HU ( lanes 5–8
    Figure Legend Snippet: Ubc9 autosumoylation negatively regulates septin sumoylation of septins in vivo . Cdc3 sumoylation in vivo is shown. Cdc3 sumoylation was analyzed in cells grown to log phase ( lanes 1–4 ) or arrested in S or G 2 /M phase with HU ( lanes 5–8

    Techniques Used: In Vivo

    Autosumoylation of Ubc9 is important for maintaining cell morphology. A , micrographs showing the cell morphology of cells with Ubc9 variants ( UBC9 WT , ubc9 K153R , ubc9 K157R and ubc9 K153R,K157R ). The arrows indicate cells with aberrant morphology. B , bar
    Figure Legend Snippet: Autosumoylation of Ubc9 is important for maintaining cell morphology. A , micrographs showing the cell morphology of cells with Ubc9 variants ( UBC9 WT , ubc9 K153R , ubc9 K157R and ubc9 K153R,K157R ). The arrows indicate cells with aberrant morphology. B , bar

    Techniques Used:

    Ubc9 is sumoylated only at Lys-153 and Lys-157. A , yeast Ubc9 wild type ( WT Ubc9), non-lysine variant ( K0 Ubc9), and lysine reversion variants ( K14,15 Ubc9 to K157 Ubc9). The positions of lysine residues, highlighted in yellow boxes , are shown on the top.
    Figure Legend Snippet: Ubc9 is sumoylated only at Lys-153 and Lys-157. A , yeast Ubc9 wild type ( WT Ubc9), non-lysine variant ( K0 Ubc9), and lysine reversion variants ( K14,15 Ubc9 to K157 Ubc9). The positions of lysine residues, highlighted in yellow boxes , are shown on the top.

    Techniques Used: Variant Assay

    Model for the regulatory role of Ubc9 autosumoylation in SUMO conjugation. SUMO conjugation in cells with or without Ubc9 autosumoylation is depicted. SUMO conjugation is achieved through successive thioester bond formation to the catalytic cysteine of
    Figure Legend Snippet: Model for the regulatory role of Ubc9 autosumoylation in SUMO conjugation. SUMO conjugation in cells with or without Ubc9 autosumoylation is depicted. SUMO conjugation is achieved through successive thioester bond formation to the catalytic cysteine of

    Techniques Used: Conjugation Assay

    Ubc9 autosumoylation negatively regulates septin sumoylation of septins in vitro . A , SUMO conjugating activity of the wild-type Ubc9 and the K153R,K157R Ubc9 variant. In vitro sumoylation was performed in the presence of partially purified Cdc3-FLAG3-His
    Figure Legend Snippet: Ubc9 autosumoylation negatively regulates septin sumoylation of septins in vitro . A , SUMO conjugating activity of the wild-type Ubc9 and the K153R,K157R Ubc9 variant. In vitro sumoylation was performed in the presence of partially purified Cdc3-FLAG3-His

    Techniques Used: In Vitro, Activity Assay, Variant Assay, Purification

    26) Product Images from "Sinorhizobium meliloti Nia is a P1B-5-ATPase expressed in the nodule during plant symbiosis and is involved in Ni and Fe transport"

    Article Title: Sinorhizobium meliloti Nia is a P1B-5-ATPase expressed in the nodule during plant symbiosis and is involved in Ni and Fe transport

    Journal: Metallomics : integrated biometal science

    doi: 10.1039/c3mt00195d

    Nia structure and genetic enviroment. (A) Topology diagram with conserved amino acids in H4 and H6 indicated. (B) Genetic environment of Nia ( Sma1163 ) cistron and determination of its constituents by RT-PCR of the intergenic sequences (1-2). Putative
    Figure Legend Snippet: Nia structure and genetic enviroment. (A) Topology diagram with conserved amino acids in H4 and H6 indicated. (B) Genetic environment of Nia ( Sma1163 ) cistron and determination of its constituents by RT-PCR of the intergenic sequences (1-2). Putative

    Techniques Used: Reverse Transcription Polymerase Chain Reaction

    27) Product Images from "Development of monoclonal antibody-based immunoassays for detection of Helicobacter pylori neutrophil-activating protein"

    Article Title: Development of monoclonal antibody-based immunoassays for detection of Helicobacter pylori neutrophil-activating protein

    Journal: Journal of immunological methods

    doi: 10.1016/j.jim.2012.06.010

    Reaction of MAb 23C8 (A) and MAb 16F4 (B) against H. pylori strain 26695 whole bacteria in immunoblotting. Approximately 10 7 bacteria undiluted (lane 1), diluted 1/4 (lane 2) or 1/16 (lane 3) were mixed 1/1 with sample buffer and loaded on criterion gel.
    Figure Legend Snippet: Reaction of MAb 23C8 (A) and MAb 16F4 (B) against H. pylori strain 26695 whole bacteria in immunoblotting. Approximately 10 7 bacteria undiluted (lane 1), diluted 1/4 (lane 2) or 1/16 (lane 3) were mixed 1/1 with sample buffer and loaded on criterion gel.

    Techniques Used:

    28) Product Images from "SpoIIE Is Necessary for Asymmetric Division, Sporulation, and Expression of ?F, ?E, and ?G but Does Not Control Solvent Production in Clostridium acetobutylicum ATCC 824 ▿ ATCC 824 ▿ ‡"

    Article Title: SpoIIE Is Necessary for Asymmetric Division, Sporulation, and Expression of ?F, ?E, and ?G but Does Not Control Solvent Production in Clostridium acetobutylicum ATCC 824 ▿ ATCC 824 ▿ ‡

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.05474-11

    Southern blot confirmation of pKOSPOIIE integration site. (A) NdeI-digested genomic DNA from C. acetobutylicum WT and SPOIIEKO hybridized with a probe for the 2nd homologous region. Lane 1, NdeI-digested SPOIIEKO DNA; lane 2, BamHI-digested pKOSPOIIE;
    Figure Legend Snippet: Southern blot confirmation of pKOSPOIIE integration site. (A) NdeI-digested genomic DNA from C. acetobutylicum WT and SPOIIEKO hybridized with a probe for the 2nd homologous region. Lane 1, NdeI-digested SPOIIEKO DNA; lane 2, BamHI-digested pKOSPOIIE;

    Techniques Used: Southern Blot

    29) Product Images from "Regulation of ciaXRH Operon Expression and Identification of the CiaR Regulon in Streptococcus mutans ▿"

    Article Title: Regulation of ciaXRH Operon Expression and Identification of the CiaR Regulon in Streptococcus mutans ▿

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.00556-10

    Effects of ciaH and ciaRH mutation and ciaR overexpression (OEciaR) on competence. The transformation efficiency of the wild type (WT) was arbitrarily assigned as 100%. The experiments were repeated 3 times, and the average values are presented.
    Figure Legend Snippet: Effects of ciaH and ciaRH mutation and ciaR overexpression (OEciaR) on competence. The transformation efficiency of the wild type (WT) was arbitrarily assigned as 100%. The experiments were repeated 3 times, and the average values are presented.

    Techniques Used: Mutagenesis, Over Expression, Transformation Assay

    30) Product Images from "Inactivation of ?F in Clostridium acetobutylicum ATCC 824 Blocks Sporulation Prior to Asymmetric Division and Abolishes ?E and ?G Protein Expression but Does Not Block Solvent Formation ▿ Protein Expression but Does Not Block Solvent Formation ▿ †"

    Article Title: Inactivation of ?F in Clostridium acetobutylicum ATCC 824 Blocks Sporulation Prior to Asymmetric Division and Abolishes ?E and ?G Protein Expression but Does Not Block Solvent Formation ▿ Protein Expression but Does Not Block Solvent Formation ▿ †

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.00088-11

    (A) Southern blot of HindIII-digested genomic DNA from C. acetobutylicum WT and FKO1 strains. Lane 1, WT DNA; lane 2, pKOSIGF; lane 3, FKO1 DNA; lane L, 2-log ladder from NEB. The membrane was exposed for 10 min. (B) Diagrams of the bands expected from the WT and FKO1 strains and of the probed region. The four short arrows represent CAC2302, CAC2303, CAC2304, and CAC2305 and are not drawn to scale.
    Figure Legend Snippet: (A) Southern blot of HindIII-digested genomic DNA from C. acetobutylicum WT and FKO1 strains. Lane 1, WT DNA; lane 2, pKOSIGF; lane 3, FKO1 DNA; lane L, 2-log ladder from NEB. The membrane was exposed for 10 min. (B) Diagrams of the bands expected from the WT and FKO1 strains and of the probed region. The four short arrows represent CAC2302, CAC2303, CAC2304, and CAC2305 and are not drawn to scale.

    Techniques Used: Southern Blot

    Integration of pKOSIGF into the C. acetobutylicum genome to disrupt the sigF gene. (A) The pKOSIGF vector with two regions of homology for sigF and the orientation of sigF in the genome. (B) A single-crossover event with pKOSIGF via the first region of homology resulted in the integration of the entire plasmid. The P ptb promoter and rho-independent terminators (RIT) are indicated. The presumable P spoIIA promoter is also indicated. (C) Primers used to confirm the integration and orientation of pKOSIGF. (D) PCR results for the WT and FKO1 strains demonstrating the integration of pKOSIGF through the first region of homology.
    Figure Legend Snippet: Integration of pKOSIGF into the C. acetobutylicum genome to disrupt the sigF gene. (A) The pKOSIGF vector with two regions of homology for sigF and the orientation of sigF in the genome. (B) A single-crossover event with pKOSIGF via the first region of homology resulted in the integration of the entire plasmid. The P ptb promoter and rho-independent terminators (RIT) are indicated. The presumable P spoIIA promoter is also indicated. (C) Primers used to confirm the integration and orientation of pKOSIGF. (D) PCR results for the WT and FKO1 strains demonstrating the integration of pKOSIGF through the first region of homology.

    Techniques Used: Plasmid Preparation, Polymerase Chain Reaction

    31) Product Images from "Functional characterization and structural modeling of synthetic polyester-degrading hydrolases from Thermomonospora curvata"

    Article Title: Functional characterization and structural modeling of synthetic polyester-degrading hydrolases from Thermomonospora curvata

    Journal: AMB Express

    doi: 10.1186/s13568-014-0044-9

    Thermal stability performance of Tcur1278 and Tcur0390. The residual hydrolytic activity was determined with (A) Tcur1278 and (B) Tcur0390 against pNPB over a period of 1 h at 50°C (solid line), 55°C (broken line) and 60°C (dotted line). Error bars indicate the standard deviation of three determinations.
    Figure Legend Snippet: Thermal stability performance of Tcur1278 and Tcur0390. The residual hydrolytic activity was determined with (A) Tcur1278 and (B) Tcur0390 against pNPB over a period of 1 h at 50°C (solid line), 55°C (broken line) and 60°C (dotted line). Error bars indicate the standard deviation of three determinations.

    Techniques Used: Activity Assay, Standard Deviation

    Molecular dynamics simulation of (A, C, E) Tcur1278 and (B, D, F) Tcur0390 polyester hydrolases. (A, B) Time courses of backbone RMSD changes during a simulation for 50 ns at 298 K (blue) and 353 K (red). (C, D) RMSF of C α atoms per amino acid residue during a simulation for 50 ns at 298 K (blue) and 353 K (red). The purple spirals and arrows at the top of the RMSF graphs indicate α-helices and β-sheets, respectively. The catalytic triad residues are shown as solid spheres. (E, F) The distance of the catalytic H208 and S130 (H-S) during a simulation for 50 ns at 298 K (blue) and 353 K (red). For a clearer view, single simulation data from three simulations are shown.
    Figure Legend Snippet: Molecular dynamics simulation of (A, C, E) Tcur1278 and (B, D, F) Tcur0390 polyester hydrolases. (A, B) Time courses of backbone RMSD changes during a simulation for 50 ns at 298 K (blue) and 353 K (red). (C, D) RMSF of C α atoms per amino acid residue during a simulation for 50 ns at 298 K (blue) and 353 K (red). The purple spirals and arrows at the top of the RMSF graphs indicate α-helices and β-sheets, respectively. The catalytic triad residues are shown as solid spheres. (E, F) The distance of the catalytic H208 and S130 (H-S) during a simulation for 50 ns at 298 K (blue) and 353 K (red). For a clearer view, single simulation data from three simulations are shown.

    Techniques Used:

    Effects of pH and temperature on the hydrolytic activity of Tcur1278 and Tcur0390. Activities of Tcur1278 and Tcur0390 against pNPB at different (A) pH and (B) reaction temperature conditions are shown as broken and solid lines, respectively. Error bars indicate the standard deviation of three determinations.
    Figure Legend Snippet: Effects of pH and temperature on the hydrolytic activity of Tcur1278 and Tcur0390. Activities of Tcur1278 and Tcur0390 against pNPB at different (A) pH and (B) reaction temperature conditions are shown as broken and solid lines, respectively. Error bars indicate the standard deviation of three determinations.

    Techniques Used: Activity Assay, Standard Deviation

    Decomposition of polyester nanoparticles by T. curvata hydrolases. PCL hydrolysis by (A) Tcur1278 and (B) Tcur0390 at 49°C; PET hydrolysis by Tcur1278 at (C) 50°C, (D) 55°C and (E) 60°C, and by Tcur0390 at (F) 50°C. The initial rates of the square roots of turbidity decrease are plotted as a function of enzyme concentration (squares and diamonds). Error bars represent the standard deviation of duplicate determinations. Fitted data (solid lines) according to Eq. ( 1 ) are also shown.
    Figure Legend Snippet: Decomposition of polyester nanoparticles by T. curvata hydrolases. PCL hydrolysis by (A) Tcur1278 and (B) Tcur0390 at 49°C; PET hydrolysis by Tcur1278 at (C) 50°C, (D) 55°C and (E) 60°C, and by Tcur0390 at (F) 50°C. The initial rates of the square roots of turbidity decrease are plotted as a function of enzyme concentration (squares and diamonds). Error bars represent the standard deviation of duplicate determinations. Fitted data (solid lines) according to Eq. ( 1 ) are also shown.

    Techniques Used: Positron Emission Tomography, Concentration Assay, Standard Deviation

    Structural modeling of Tcur1278 and Tcur0390 polyester hydrolases. Homology modeling was performed with the Phyre2 web server (Kelley and Sternberg [ 2009 ]). The catalytic triad of (A) Tcur1278 and (B) Tcur0390 is formed by S130, D176 and H208. The 2PET model substrate was docked using GOLD 5.1 with its central ester bond constrained between 2.7 and 3.1 Å in the oxyanion hole formed by the main chain NH groups of F62 and M131 (broken yellow lines). The hydrogen bonds stabilizing the tetrahedral intermediate formed during the catalytic reaction are shown as broken lines in blue. The backbone structures are shown as gray cartoons. The electrostatic surface properties of Tcur1278 (C) and Tcur0390 (D) are shown with negatively charged residues in red, positively charged residues in blue and neutral residues in white/gray, respectively. The lipophilic surface properties of Tcur1278 (E) and Tcur0390 (F) are shown with hydrophilic residues in pink and hydrophobic residues in bright green, respectively. The docked 2PET model substrate is shown in cyan.
    Figure Legend Snippet: Structural modeling of Tcur1278 and Tcur0390 polyester hydrolases. Homology modeling was performed with the Phyre2 web server (Kelley and Sternberg [ 2009 ]). The catalytic triad of (A) Tcur1278 and (B) Tcur0390 is formed by S130, D176 and H208. The 2PET model substrate was docked using GOLD 5.1 with its central ester bond constrained between 2.7 and 3.1 Å in the oxyanion hole formed by the main chain NH groups of F62 and M131 (broken yellow lines). The hydrogen bonds stabilizing the tetrahedral intermediate formed during the catalytic reaction are shown as broken lines in blue. The backbone structures are shown as gray cartoons. The electrostatic surface properties of Tcur1278 (C) and Tcur0390 (D) are shown with negatively charged residues in red, positively charged residues in blue and neutral residues in white/gray, respectively. The lipophilic surface properties of Tcur1278 (E) and Tcur0390 (F) are shown with hydrophilic residues in pink and hydrophobic residues in bright green, respectively. The docked 2PET model substrate is shown in cyan.

    Techniques Used:

    32) Product Images from "Phylogenetic Analysis of Glucosyltransferases and Implications for the Coevolution of Mutans Streptococci with Their Mammalian Hosts"

    Article Title: Phylogenetic Analysis of Glucosyltransferases and Implications for the Coevolution of Mutans Streptococci with Their Mammalian Hosts

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0056305

    Models of the 3D structure of the catalytic domain of S. mutans Gtfs. Blue: amino acids under negative selection (side chains shown). Yellow: active site. Magenta: Gtf-P1 region [69] . Green sphere: Ca 2+ ion. Right panels: Detail of a different view of the region surrounding the active site of GtfB and GtfC.
    Figure Legend Snippet: Models of the 3D structure of the catalytic domain of S. mutans Gtfs. Blue: amino acids under negative selection (side chains shown). Yellow: active site. Magenta: Gtf-P1 region [69] . Green sphere: Ca 2+ ion. Right panels: Detail of a different view of the region surrounding the active site of GtfB and GtfC.

    Techniques Used: Selection

    Site specific profile of type I functional divergence posterior probability. Logos are shown for positions predicted to be critical for type I functional divergence between GtfB and GtfC (cutoff P > 0.85). Residues are color coded by biochemical property and heights represent their relative frequency at each site. The Gtf protein domains are represented below the graph. I) signal peptide, II) N-terminal variable region, III) catalytic domain, IV) glucan binding domain.
    Figure Legend Snippet: Site specific profile of type I functional divergence posterior probability. Logos are shown for positions predicted to be critical for type I functional divergence between GtfB and GtfC (cutoff P > 0.85). Residues are color coded by biochemical property and heights represent their relative frequency at each site. The Gtf protein domains are represented below the graph. I) signal peptide, II) N-terminal variable region, III) catalytic domain, IV) glucan binding domain.

    Techniques Used: Functional Assay, Binding Assay

    33) Product Images from "Myeloid-Derived Suppressor Cells Produce IL-10 to Elicit DNMT3b-Dependent IRF8 Silencing to Promote Colitis-Associated Colon Tumorigenesis"

    Article Title: Myeloid-Derived Suppressor Cells Produce IL-10 to Elicit DNMT3b-Dependent IRF8 Silencing to Promote Colitis-Associated Colon Tumorigenesis

    Journal: Cell reports

    doi: 10.1016/j.celrep.2018.11.050

    The Irf8 Promoter Is Hypermethylated in Chronic Inflammation-Induced Colon Tumor (A) The mouse Irf8 promoter structure. The CpG islands are indicated by blue, transcription initiation site is indicated by +1. The numbers above the bar indicate the nucleotide location relative to the Irf8 transcription initiation site. Bottom: genomic DNA was extracted from the colon tissues of normal tumor-free mice (n = 3) and the colon tumor tissues of AOM-DSS-treated mice (n = 3) and modified with bisulfite. The modified genomic DNA was then amplified with bisulfite-modified DNA-specific primers to amplify a CpG island region, as indicated under the CpG island (–348 to +156). The amplified DNA fragments were cloned and sequenced. Each circle represents a CpG dinucleotide. Open circles indicate unmethylated CpG, and closed circles represent methylated CpG. (B) The bisulfite-modified genomic DNA as in (A) was also analyzed by methylation-specific (MS)-PCR. U, unmethylated; M, methylated. (C) Bisulfite-sequencing analysis of the CpG island region in the Irf8 promoter of colon epithelial CCD841 and colon carcinoma HCT116 cells. Each circle represents a CpG dinucleotide. Open circles indicate unmethylated CpG, and closed circles represent methylated CpG. (D) The Irf8 promoter DNA methylation datasets of normal human colon tissues and colorectal carcinoma tissues were extracted from TCGA database and compared. (E) MS-PCR analysis of the Irf8 promoter region in WT, DNMT1 −/− , DNMT3b −/− and DKO of HCT116 cells. U, unmethylated; M, methylated. (F) RNA was extracted from WT, DNMT1 −/− , DNMT3b −/− , and DKO of HCT116 cells and analyzed for IRF8 mRNA expression levels by semiquantitative RT-PCR (right top) and qPCR (left) using β-actin as an internal control. Bar, SD. Bottom right: the IRF8 protein level was analyzed by western blotting using IRF8-specific antibody. β-Actin was used as a normalization control.
    Figure Legend Snippet: The Irf8 Promoter Is Hypermethylated in Chronic Inflammation-Induced Colon Tumor (A) The mouse Irf8 promoter structure. The CpG islands are indicated by blue, transcription initiation site is indicated by +1. The numbers above the bar indicate the nucleotide location relative to the Irf8 transcription initiation site. Bottom: genomic DNA was extracted from the colon tissues of normal tumor-free mice (n = 3) and the colon tumor tissues of AOM-DSS-treated mice (n = 3) and modified with bisulfite. The modified genomic DNA was then amplified with bisulfite-modified DNA-specific primers to amplify a CpG island region, as indicated under the CpG island (–348 to +156). The amplified DNA fragments were cloned and sequenced. Each circle represents a CpG dinucleotide. Open circles indicate unmethylated CpG, and closed circles represent methylated CpG. (B) The bisulfite-modified genomic DNA as in (A) was also analyzed by methylation-specific (MS)-PCR. U, unmethylated; M, methylated. (C) Bisulfite-sequencing analysis of the CpG island region in the Irf8 promoter of colon epithelial CCD841 and colon carcinoma HCT116 cells. Each circle represents a CpG dinucleotide. Open circles indicate unmethylated CpG, and closed circles represent methylated CpG. (D) The Irf8 promoter DNA methylation datasets of normal human colon tissues and colorectal carcinoma tissues were extracted from TCGA database and compared. (E) MS-PCR analysis of the Irf8 promoter region in WT, DNMT1 −/− , DNMT3b −/− and DKO of HCT116 cells. U, unmethylated; M, methylated. (F) RNA was extracted from WT, DNMT1 −/− , DNMT3b −/− , and DKO of HCT116 cells and analyzed for IRF8 mRNA expression levels by semiquantitative RT-PCR (right top) and qPCR (left) using β-actin as an internal control. Bar, SD. Bottom right: the IRF8 protein level was analyzed by western blotting using IRF8-specific antibody. β-Actin was used as a normalization control.

    Techniques Used: Mouse Assay, Modification, Amplification, Clone Assay, Methylation, Mass Spectrometry, Polymerase Chain Reaction, Methylation Sequencing, DNA Methylation Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Western Blot

    IL-10 Induces the Activation of STAT3 that Binds to the dnmt1 and dnmt3b Promoters in Colon Epithelial and Carcinoma Cells (A) WT C57BL/6 mice were treated with the 2% DSS-water cycle, as described in STAR Methods . Colon tissues were collected from mice at the indicated time points and analyzed by western blotting for STAT1 and STAT3 protein levels. β-Actin was used as a normalization control. (B) CCD841 and HT29 cells were treated with recombinant IL-10 (100 ng/mL) for 2 hr and analyzed for the indicated proteins by western blotting. (C) Top: structure of the Dnmt1 promoter region. The number below the bar indicates nucleotide locations relative to the Dnmt1 transcription initiation site. The ChIP PCR primer regions are indicated under the bar. Bottom: CCD841 and HT29 cells were stimulated with recombinant IL-10 protein (100 ng/mL) for 16 hr, then analyzed by ChIP using immunoglobulin G (IgG) control antibody and pSTAT3-specific antibody, respectively, followed by qPCR analysis with Dnmt1 promoter DNA-specific PCR primers, as shown at top. Input DNA was used as a normalization control. The input of each ChIP primer set was arbitrarily set at 1, and the pSTAT3 was normalized to the input DNA level. Column, mean; bar, SD. (D) Top: structure of the Dnmt3b promoter region. The number below the bar indicates nucleotide locations relative to the Dnmt3b transcription initiation site. The ChIP PCR primer regions are indicated under the bar. Bottom: CCD841 and HT29 cells were stimulated with recombinant IL-10 protein (100 ng/mL) for 16 hr, then analyzed by ChIP using IgG control antibody and pSTAT3-specific antibody, respectively, followed by qPCR analysis with Dnmt3b promoter DNA-specific PCR primers, as shown at top. Input DNA was used as a normalization control. The input of each ChIP primer set was arbitrarily set at 1, and the pSTAT3 was normalized to the input DNA level. Column, mean; bar, SD. (E and F) The human DNMT1 (E) and DNMT3b (F) promoter DNA fragments were amplified by PCR from the two indicated regions (top: P1 and P2 for DNMT1 , and P3 and P4 for DNMT3b ) and cloned to the pGL3 vector. pGL3 vectors containing the P1, P2, P3, or P4 DNA fragments were transiently transfected to CCD841 and HT29 cells, respectively, overnight. Cells were either untreated (control) or treated with IL-10 (100 ng/mL) for 4 hr. Cells were lysated and analyzed for luciferase activity. Bar, SD.
    Figure Legend Snippet: IL-10 Induces the Activation of STAT3 that Binds to the dnmt1 and dnmt3b Promoters in Colon Epithelial and Carcinoma Cells (A) WT C57BL/6 mice were treated with the 2% DSS-water cycle, as described in STAR Methods . Colon tissues were collected from mice at the indicated time points and analyzed by western blotting for STAT1 and STAT3 protein levels. β-Actin was used as a normalization control. (B) CCD841 and HT29 cells were treated with recombinant IL-10 (100 ng/mL) for 2 hr and analyzed for the indicated proteins by western blotting. (C) Top: structure of the Dnmt1 promoter region. The number below the bar indicates nucleotide locations relative to the Dnmt1 transcription initiation site. The ChIP PCR primer regions are indicated under the bar. Bottom: CCD841 and HT29 cells were stimulated with recombinant IL-10 protein (100 ng/mL) for 16 hr, then analyzed by ChIP using immunoglobulin G (IgG) control antibody and pSTAT3-specific antibody, respectively, followed by qPCR analysis with Dnmt1 promoter DNA-specific PCR primers, as shown at top. Input DNA was used as a normalization control. The input of each ChIP primer set was arbitrarily set at 1, and the pSTAT3 was normalized to the input DNA level. Column, mean; bar, SD. (D) Top: structure of the Dnmt3b promoter region. The number below the bar indicates nucleotide locations relative to the Dnmt3b transcription initiation site. The ChIP PCR primer regions are indicated under the bar. Bottom: CCD841 and HT29 cells were stimulated with recombinant IL-10 protein (100 ng/mL) for 16 hr, then analyzed by ChIP using IgG control antibody and pSTAT3-specific antibody, respectively, followed by qPCR analysis with Dnmt3b promoter DNA-specific PCR primers, as shown at top. Input DNA was used as a normalization control. The input of each ChIP primer set was arbitrarily set at 1, and the pSTAT3 was normalized to the input DNA level. Column, mean; bar, SD. (E and F) The human DNMT1 (E) and DNMT3b (F) promoter DNA fragments were amplified by PCR from the two indicated regions (top: P1 and P2 for DNMT1 , and P3 and P4 for DNMT3b ) and cloned to the pGL3 vector. pGL3 vectors containing the P1, P2, P3, or P4 DNA fragments were transiently transfected to CCD841 and HT29 cells, respectively, overnight. Cells were either untreated (control) or treated with IL-10 (100 ng/mL) for 4 hr. Cells were lysated and analyzed for luciferase activity. Bar, SD.

    Techniques Used: Activation Assay, Mouse Assay, Western Blot, Recombinant, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Amplification, Clone Assay, Plasmid Preparation, Transfection, Luciferase, Activity Assay

    IL-10 Upregulates DNMT1 and DNMT3b Expression in Colon Epithelial and Carcinoma Cells (A) Colonic epithelial CCD841 and colon carcinoma HT29 cells were treated with recombinant IL-10 at the indicated doses for 24 hr and analyzed for DNMT1 and DNMT3b mRNA expression levels by semiquantitative RT-PCR (top) and qPCR (bottom) using β-actin as an internal control. Bar, SD. (B) CCD841 and HT29 cells were treated with recombinant IL-10 (100 ng/mL) for 24 hr and analyzed by western blotting analysis of DNMT1, DNMT3b (left), and IRF8 (right) protein levels. (C) WT (n = 4) and IL-10 KO (n = 5) mice were treated with the DSS-water cycle, as described in STAR Methods for 28 days. Colon tissues were collected and analyzed by western blotting for DNMT1 and DNMT3b protein levels. (D) WT (n = 3) and IL-10 KO (n = 3) mice were treated with the 2% DSS-water cycle, as described in STAR Methods . Colon tissues were collected at day 28 and analyzed by qPCR for the IRF8 mRNA level with β-actin as an internal control. Bar, SD. (E) The pGL3 vector containing the human IRF8 promoter was treated with methylase in vitro and transfected to CCD841 cells overnight. Cells were lysated and analyzed for luciferase activity, as described in STAR Methods .
    Figure Legend Snippet: IL-10 Upregulates DNMT1 and DNMT3b Expression in Colon Epithelial and Carcinoma Cells (A) Colonic epithelial CCD841 and colon carcinoma HT29 cells were treated with recombinant IL-10 at the indicated doses for 24 hr and analyzed for DNMT1 and DNMT3b mRNA expression levels by semiquantitative RT-PCR (top) and qPCR (bottom) using β-actin as an internal control. Bar, SD. (B) CCD841 and HT29 cells were treated with recombinant IL-10 (100 ng/mL) for 24 hr and analyzed by western blotting analysis of DNMT1, DNMT3b (left), and IRF8 (right) protein levels. (C) WT (n = 4) and IL-10 KO (n = 5) mice were treated with the DSS-water cycle, as described in STAR Methods for 28 days. Colon tissues were collected and analyzed by western blotting for DNMT1 and DNMT3b protein levels. (D) WT (n = 3) and IL-10 KO (n = 3) mice were treated with the 2% DSS-water cycle, as described in STAR Methods . Colon tissues were collected at day 28 and analyzed by qPCR for the IRF8 mRNA level with β-actin as an internal control. Bar, SD. (E) The pGL3 vector containing the human IRF8 promoter was treated with methylase in vitro and transfected to CCD841 cells overnight. Cells were lysated and analyzed for luciferase activity, as described in STAR Methods .

    Techniques Used: Expressing, Recombinant, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Western Blot, Mouse Assay, Plasmid Preparation, In Vitro, Transfection, Luciferase, Activity Assay

    34) Product Images from "Characterization of an antimicrobial and phytotoxic ribonuclease secreted by the fungal wheat pathogen Zymoseptoria tritici"

    Article Title: Characterization of an antimicrobial and phytotoxic ribonuclease secreted by the fungal wheat pathogen Zymoseptoria tritici

    Journal: The New Phytologist

    doi: 10.1111/nph.14786

    Zt6 is toxic to bacteria and yeasts, but not to Zymoseptoria tritici . Recombinant Zt6 ∆19–40 and the ribotoxin restrictocin incubated with (a) Pichia pastoris , (b) Saccharomyces cerevisiae , (c) Escherichia coli and (d) Z. tritici at 20 μM concentration. Bars represent mean ± SE .
    Figure Legend Snippet: Zt6 is toxic to bacteria and yeasts, but not to Zymoseptoria tritici . Recombinant Zt6 ∆19–40 and the ribotoxin restrictocin incubated with (a) Pichia pastoris , (b) Saccharomyces cerevisiae , (c) Escherichia coli and (d) Z. tritici at 20 μM concentration. Bars represent mean ± SE .

    Techniques Used: Recombinant, Incubation, Concentration Assay

    35) Product Images from "The Phage Lysin PlySs2 Decolonizes Streptococcus suis from Murine Intranasal Mucosa"

    Article Title: The Phage Lysin PlySs2 Decolonizes Streptococcus suis from Murine Intranasal Mucosa

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0169180

    S . suis 7997 and S735 did not develop resistance to PlySs2 in vitro . S . suis strain S735 or S . suis strain 7997 grew in media containing 1/32× (3.13%) to 4× (400%) the MIC of PlySs2 or gentamicin over 8 days. Comparing the MICs of PlySs2 after each day to the initial MIC of PlySs2 for each strain determined resistance. Neither developed resistance to PlySs2. Both S . suis strain S735 and S . suis strain 7997 developed resistance to the positive control, gentamicin. The fluctuation observed in this assay was +/- 1x MIC.
    Figure Legend Snippet: S . suis 7997 and S735 did not develop resistance to PlySs2 in vitro . S . suis strain S735 or S . suis strain 7997 grew in media containing 1/32× (3.13%) to 4× (400%) the MIC of PlySs2 or gentamicin over 8 days. Comparing the MICs of PlySs2 after each day to the initial MIC of PlySs2 for each strain determined resistance. Neither developed resistance to PlySs2. Both S . suis strain S735 and S . suis strain 7997 developed resistance to the positive control, gentamicin. The fluctuation observed in this assay was +/- 1x MIC.

    Techniques Used: In Vitro, Positive Control

    PlySs2 and gentamicin may act additively to reduce S . suis in vivo . PlySs2 removed S . suis from the murine intranasal mucosa. CD-1 ® mice were nasally colonized with the pathogenic S . suis strain 7997. Twenty-four hours after colonization, in each nostril, mice received 10 μl of either 50 mM PB, pH 7.4 (buffer C), 5 mg/ml PlySs2 in buffer C, 5 mg/ml gentamicin in buffer C, or a combination of 2.5 mg/ml PlySs2 and 2.5 mg/ml gentamicin in buffer C. S . suis CFU counts were calculated for the nasal passage of each mouse.
    Figure Legend Snippet: PlySs2 and gentamicin may act additively to reduce S . suis in vivo . PlySs2 removed S . suis from the murine intranasal mucosa. CD-1 ® mice were nasally colonized with the pathogenic S . suis strain 7997. Twenty-four hours after colonization, in each nostril, mice received 10 μl of either 50 mM PB, pH 7.4 (buffer C), 5 mg/ml PlySs2 in buffer C, 5 mg/ml gentamicin in buffer C, or a combination of 2.5 mg/ml PlySs2 and 2.5 mg/ml gentamicin in buffer C. S . suis CFU counts were calculated for the nasal passage of each mouse.

    Techniques Used: Activated Clotting Time Assay, In Vivo, Mouse Assay

    PlySs2 displayed activity against almost all strains of S . suis . Bacteria in logarithmic growth were exposed to 32 μg/ml PlySs2 for 30 minutes in PB (for 60-minute readings, see S3 Fig ). The activity was measured by OD 600 reduction. To normalize and combine values from multiple tests, the final OD 600 of the treated samples was divided by the final OD 600 of the untreated samples. An OD 600 ratio of 1.0 indicates no lysis, while an OD 600 ratio of ~0.02 indicates complete lysis.
    Figure Legend Snippet: PlySs2 displayed activity against almost all strains of S . suis . Bacteria in logarithmic growth were exposed to 32 μg/ml PlySs2 for 30 minutes in PB (for 60-minute readings, see S3 Fig ). The activity was measured by OD 600 reduction. To normalize and combine values from multiple tests, the final OD 600 of the treated samples was divided by the final OD 600 of the untreated samples. An OD 600 ratio of 1.0 indicates no lysis, while an OD 600 ratio of ~0.02 indicates complete lysis.

    Techniques Used: Activity Assay, Lysis

    PlySs2 was bactericidal to nearly all strains of S . suis . Bacteria were grown to log-phase. After exposure to 64 μg/ml PlySs2 in buffer A for 60 min in 96-well plates, bacteria were serially diluted and plated to BHI agar for CFU enumeration. The CFU numbers of most S . suis strains dropped by 5 to 6 logs after PlySs2 treatment including the type strain S735 and the pathogenic strains 10 and 7997. Death (log fold kill) was calculated as -log[(CFUs in the test condition) ÷ (CFUs in the control condition)].
    Figure Legend Snippet: PlySs2 was bactericidal to nearly all strains of S . suis . Bacteria were grown to log-phase. After exposure to 64 μg/ml PlySs2 in buffer A for 60 min in 96-well plates, bacteria were serially diluted and plated to BHI agar for CFU enumeration. The CFU numbers of most S . suis strains dropped by 5 to 6 logs after PlySs2 treatment including the type strain S735 and the pathogenic strains 10 and 7997. Death (log fold kill) was calculated as -log[(CFUs in the test condition) ÷ (CFUs in the control condition)].

    Techniques Used:

    36) Product Images from "Use of an EZ-Tn5-Based Random Mutagenesis System to Identify a Novel Toxin Regulatory Locus in Clostridium perfringens Strain 13"

    Article Title: Use of an EZ-Tn5-Based Random Mutagenesis System to Identify a Novel Toxin Regulatory Locus in Clostridium perfringens Strain 13

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0006232

    Modification of the EZ-Tn 5 -encoding vector for random mutagenesis in C. perfringens . To allow selection of C. perfringens transformants after electroporation with the EZ-Tn 5 transposon, a C. perfringens erythromycin resistance determinant ( erm ) was cloned into the multiple cloning site in the Epicentre EZ-TN 5 -encoding pMOD-2 vector creating pJVTN5. This plasmid also contains PvuII-recognized sequences flanking the mosaic end (ME) sites, which are specifically recognized by the EZ-Tn 5 transposase.
    Figure Legend Snippet: Modification of the EZ-Tn 5 -encoding vector for random mutagenesis in C. perfringens . To allow selection of C. perfringens transformants after electroporation with the EZ-Tn 5 transposon, a C. perfringens erythromycin resistance determinant ( erm ) was cloned into the multiple cloning site in the Epicentre EZ-TN 5 -encoding pMOD-2 vector creating pJVTN5. This plasmid also contains PvuII-recognized sequences flanking the mosaic end (ME) sites, which are specifically recognized by the EZ-Tn 5 transposase.

    Techniques Used: Modification, Plasmid Preparation, Mutagenesis, Selection, Electroporation, Clone Assay

    37) Product Images from "Characterization of Endogenous Plasmids from Lactobacillus salivarius UCC118 "

    Article Title: Characterization of Endogenous Plasmids from Lactobacillus salivarius UCC118

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.02631-07

    Annotation of L. salivarius UCC118 plasmids pSF118-20 and pSF118-44.
    Figure Legend Snippet: Annotation of L. salivarius UCC118 plasmids pSF118-20 and pSF118-44.

    Techniques Used:

    Function of the TA system on pSF118-20. (A) Contribution of the TA system to pLS203 maintenance in L. salivarius LS201, L. plantarum NCIMB8826, and L. fermentum DSM20055. Plasmid retention after 100 generations of growth for each strain was compared with
    Figure Legend Snippet: Function of the TA system on pSF118-20. (A) Contribution of the TA system to pLS203 maintenance in L. salivarius LS201, L. plantarum NCIMB8826, and L. fermentum DSM20055. Plasmid retention after 100 generations of growth for each strain was compared with

    Techniques Used: Plasmid Preparation

    Plasmid profiles of pSF118-20- and pSF118-44-related replication regions in 27 L. salivarius strains. Southern hybridization of nuclease S1-treated genomic DNA of 27 L. salivarius strains with the pSF118-20 repA probe (A) and the pSF118-44 repA probe
    Figure Legend Snippet: Plasmid profiles of pSF118-20- and pSF118-44-related replication regions in 27 L. salivarius strains. Southern hybridization of nuclease S1-treated genomic DNA of 27 L. salivarius strains with the pSF118-20 repA probe (A) and the pSF118-44 repA probe

    Techniques Used: Plasmid Preparation, Hybridization

    Characterization of pLS208 transconjugants. (A) Plasmid profiles of two representative transconjugants obtained from mating between L. plantarum NCIMB8826(pLS208) and either L. fermentum DSM20055 (pNZ8048) (T1) or L. salivarius JCM1045(pNZ8048) (T2).
    Figure Legend Snippet: Characterization of pLS208 transconjugants. (A) Plasmid profiles of two representative transconjugants obtained from mating between L. plantarum NCIMB8826(pLS208) and either L. fermentum DSM20055 (pNZ8048) (T1) or L. salivarius JCM1045(pNZ8048) (T2).

    Techniques Used: Plasmid Preparation

    Analysis of the replication mechanism of pSF118-20. (A) Cell lysates of L. salivarius LS201 strains harboring pLS203 (an E. coli-Lactobacillus shuttle vector containing the replication origin of pSF118-20) or pVE6007 (a rolling-circle replication plasmid
    Figure Legend Snippet: Analysis of the replication mechanism of pSF118-20. (A) Cell lysates of L. salivarius LS201 strains harboring pLS203 (an E. coli-Lactobacillus shuttle vector containing the replication origin of pSF118-20) or pVE6007 (a rolling-circle replication plasmid

    Techniques Used: Plasmid Preparation

    Growth of L. salivarius LS201(pLS210), L. plantarum NCIMB8826(pLS210), and L. fermentum DSM20055(pLS210) and the concomitant detection of bioluminescence (A, B). (A) Lane 1, L. salivarius LS201(pLS210); lane 2, L. plantarum NCIMB8826(pLS210); lane 3,
    Figure Legend Snippet: Growth of L. salivarius LS201(pLS210), L. plantarum NCIMB8826(pLS210), and L. fermentum DSM20055(pLS210) and the concomitant detection of bioluminescence (A, B). (A) Lane 1, L. salivarius LS201(pLS210); lane 2, L. plantarum NCIMB8826(pLS210); lane 3,

    Techniques Used:

    Comparison of the conjugal transfer ( tra ) regions in selected plasmids. pMP118, the megaplasmid from L. salivarius ); pMRC01, the conjugative plasmid from L. lactis ); pWCFS103, the conjugative plasmid from L. plantarum NCIMB8826 (
    Figure Legend Snippet: Comparison of the conjugal transfer ( tra ) regions in selected plasmids. pMP118, the megaplasmid from L. salivarius ); pMRC01, the conjugative plasmid from L. lactis ); pWCFS103, the conjugative plasmid from L. plantarum NCIMB8826 (

    Techniques Used: Plasmid Preparation

    Confirmation of the curing of pSF118-20 from L. salivarius UCC118. (A) Southern hybridization analysis of L. salivarius UCC118 (lane 1) and its cured derivative LS201 (lane 2). Genomic DNA was either undigested or digested with EcoRV, and then blots were
    Figure Legend Snippet: Confirmation of the curing of pSF118-20 from L. salivarius UCC118. (A) Southern hybridization analysis of L. salivarius UCC118 (lane 1) and its cured derivative LS201 (lane 2). Genomic DNA was either undigested or digested with EcoRV, and then blots were

    Techniques Used: Hybridization

    38) Product Images from "Coxiella burnetii Effector Proteins That Localize to the Parasitophorous Vacuole Membrane Promote Intracellular Replication"

    Article Title: Coxiella burnetii Effector Proteins That Localize to the Parasitophorous Vacuole Membrane Promote Intracellular Replication

    Journal: Infection and Immunity

    doi: 10.1128/IAI.02763-14

    Four C. burnetii Dot/Icm T4BSS substrates localize to the PV membrane. (A) Representative confocal fluorescence micrographs of C. burnetii -infected HeLa cells ectopically expressing CvpB, CvpC, CvpD, or CvpE N-terminally fused to GFP. At 72 h postinfection,
    Figure Legend Snippet: Four C. burnetii Dot/Icm T4BSS substrates localize to the PV membrane. (A) Representative confocal fluorescence micrographs of C. burnetii -infected HeLa cells ectopically expressing CvpB, CvpC, CvpD, or CvpE N-terminally fused to GFP. At 72 h postinfection,

    Techniques Used: Fluorescence, Infection, Expressing

    C. burnetii Δ cvp mutants exhibit defects in intracellular growth and PV biogenesis. (A) Replication of wild-type C. burnetii and Δ cvp mutants. Fold increases in bacterial GE at 6 days postinfection of THP-1 macrophages relative to the
    Figure Legend Snippet: C. burnetii Δ cvp mutants exhibit defects in intracellular growth and PV biogenesis. (A) Replication of wild-type C. burnetii and Δ cvp mutants. Fold increases in bacterial GE at 6 days postinfection of THP-1 macrophages relative to the

    Techniques Used:

    Coinfection with wild-type C. burnetii rescues growth of Δ cvpB and Δ cvpC mutants. THP-1 macrophages were infected with the Δ cvpB or Δ cvpC mutant alone or coinfected with the Δ cvpB or Δ cvpC mutant and wild-type
    Figure Legend Snippet: Coinfection with wild-type C. burnetii rescues growth of Δ cvpB and Δ cvpC mutants. THP-1 macrophages were infected with the Δ cvpB or Δ cvpC mutant alone or coinfected with the Δ cvpB or Δ cvpC mutant and wild-type

    Techniques Used: Infection, Mutagenesis

    Identification of C. burnetii T4BSS effector proteins.
    Figure Legend Snippet: Identification of C. burnetii T4BSS effector proteins.

    Techniques Used:

    Genetic complementation of the Δ cvpD and Δ cvpE mutants rescues intracellular growth and PV biogenesis. (A) Replication of wild-type C. burnetii and complemented Δ cvp mutants. Fold increases in bacterial GE at 6 days postinfection
    Figure Legend Snippet: Genetic complementation of the Δ cvpD and Δ cvpE mutants rescues intracellular growth and PV biogenesis. (A) Replication of wild-type C. burnetii and complemented Δ cvp mutants. Fold increases in bacterial GE at 6 days postinfection

    Techniques Used:

    C. burnetii translocates 13 proteins via its Dot/Icm T4BSS. Cytosolic concentrations of cAMP were determined after infection of THP-1 macrophages for 48 h with wild-type C. burnetii or a Δ dotA mutant expressing CyaA fusions to candidate Dot/Icm
    Figure Legend Snippet: C. burnetii translocates 13 proteins via its Dot/Icm T4BSS. Cytosolic concentrations of cAMP were determined after infection of THP-1 macrophages for 48 h with wild-type C. burnetii or a Δ dotA mutant expressing CyaA fusions to candidate Dot/Icm

    Techniques Used: Infection, Mutagenesis, Expressing

    39) Product Images from "Complex two-component signaling regulates the general stress response in Alphaproteobacteria"

    Article Title: Complex two-component signaling regulates the general stress response in Alphaproteobacteria

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

    doi: 10.1073/pnas.1410095111

    Summary of the current model of GSR regulation in S. melonis Fr1 (details are provided in the main text). Colored arrows indicate activating stress conditions for kinases. Black arrows and bars represent activating and inhibitory relationships, respectively.
    Figure Legend Snippet: Summary of the current model of GSR regulation in S. melonis Fr1 (details are provided in the main text). Colored arrows indicate activating stress conditions for kinases. Black arrows and bars represent activating and inhibitory relationships, respectively.

    Techniques Used:

    40) Product Images from "Genome-Wide Transposon Mutagenesis of Borrelia burgdorferi for Identification of Phenotypic Mutants"

    Article Title: Genome-Wide Transposon Mutagenesis of Borrelia burgdorferi for Identification of Phenotypic Mutants

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.70.10.5973-5979.2004

    Transposon mutagenesis system for B. burgdorferi . Suicide vector pMarGent was electroporated into competent B. burgdorferi cells, allowing transient expression of the Himar1 transposase (1). After transposition, the Himar1 gene remains on a DNA fragment that is presumably degraded by intracellular nucleases (2). Mutants were selected in the presence of gentamicin, and those with desired phenotypes were transferred to liquid culture for DNA isolation. The B. burgdorferi DNA flanking the transposon insertion site was recovered by digestion with HindIII, an enzyme that does not cut within the transposon (3 and 4); self-ligation; and transformation into E. coli (5). Purified plasmid DNA was then isolated from E. coli clones and sequenced. IR, inverted repeat.
    Figure Legend Snippet: Transposon mutagenesis system for B. burgdorferi . Suicide vector pMarGent was electroporated into competent B. burgdorferi cells, allowing transient expression of the Himar1 transposase (1). After transposition, the Himar1 gene remains on a DNA fragment that is presumably degraded by intracellular nucleases (2). Mutants were selected in the presence of gentamicin, and those with desired phenotypes were transferred to liquid culture for DNA isolation. The B. burgdorferi DNA flanking the transposon insertion site was recovered by digestion with HindIII, an enzyme that does not cut within the transposon (3 and 4); self-ligation; and transformation into E. coli (5). Purified plasmid DNA was then isolated from E. coli clones and sequenced. IR, inverted repeat.

    Techniques Used: Mutagenesis, Plasmid Preparation, Expressing, DNA Extraction, Ligation, Transformation Assay, Purification, Isolation, Clone Assay

    Related Articles

    Clone Assay:

    Article Title: Streamlined ex vivo and in vivo genome editing in mouse embryos using recombinant adeno-associated viruses
    Article Snippet: .. Purified PCR products were cloned into the pCRTM -Blunt II-TOPO vector using Zero Blunt TOPO PCR Cloning Kit (Thermo Fisher Sci. .. Cat. No. K280002), and used to transform DH5α Escherichia coli bacteria.

    Article Title: Streamlined ex vivo and in vivo genome editing in mouse embryos using recombinant adeno-associated viruses
    Article Snippet: .. Purified PCR product was cloned into the pCRTM -Blunt II-TOPO vector using Zero Blunt TOPO PCR Cloning Kit (Thermo Fisher Sci. .. Cat. No. K280002) and sequenced using the Sanger method.

    Article Title: Elimination of 01/A′–A0 pre-rRNA processing by-product in human cells involves cooperative action of two nuclear exosome-associated nucleases: RRP6 and DIS3
    Article Snippet: .. The final PCR products were cloned with the use of Zero Blunt Topo PCR Cloning Kit (Invitrogen), and the inserts were sequenced using M13 Forward (−20) and M13 Reverse oligonucleotides, following recommendations of the manufacturer. .. High-throughput analysis of the RNA 3′-ends was performed essentially as described previously , with some modifications listed hereafter, using 2 µg of total RNA as a starting material.

    Article Title: CRISPR/Cas9 Beta-globin Gene Targeting in Human Hematopoietic Stem Cells
    Article Snippet: .. The PCR product was gel-purified and re-amplified using a nested primer set ( HBB _nested_fw: 5’-GAAGATATGCTTAGAACCGAGG-3’ and HBB _nested_rw: 5’-CCACATGCCCAGTTTCTATTGG-3’) to create a 685bp PCR amplicon (see ) that was gel-purified and cloned into a TOPO plasmid using the Zero Blunt TOPO PCR Cloning Kit (Life Technologies) according to the manufacturer's protocol. .. TOPO reactions were transformed into XL-1 Blue competent cells, plated on kanamycin-containing agar plates, and single colonies were sequenced by McLab (South San Francisco, CA, USA) by rolling circle amplification followed by sequencing using the following primer: 5′-GAAGATATGCTTAGAACCGAGG-3′.

    Amplification:

    Article Title: CRISPR/Cas9 Beta-globin Gene Targeting in Human Hematopoietic Stem Cells
    Article Snippet: .. The PCR product was gel-purified and re-amplified using a nested primer set ( HBB _nested_fw: 5’-GAAGATATGCTTAGAACCGAGG-3’ and HBB _nested_rw: 5’-CCACATGCCCAGTTTCTATTGG-3’) to create a 685bp PCR amplicon (see ) that was gel-purified and cloned into a TOPO plasmid using the Zero Blunt TOPO PCR Cloning Kit (Life Technologies) according to the manufacturer's protocol. .. TOPO reactions were transformed into XL-1 Blue competent cells, plated on kanamycin-containing agar plates, and single colonies were sequenced by McLab (South San Francisco, CA, USA) by rolling circle amplification followed by sequencing using the following primer: 5′-GAAGATATGCTTAGAACCGAGG-3′.

    Purification:

    Article Title: Streamlined ex vivo and in vivo genome editing in mouse embryos using recombinant adeno-associated viruses
    Article Snippet: .. Purified PCR products were cloned into the pCRTM -Blunt II-TOPO vector using Zero Blunt TOPO PCR Cloning Kit (Thermo Fisher Sci. .. Cat. No. K280002), and used to transform DH5α Escherichia coli bacteria.

    Article Title: Streamlined ex vivo and in vivo genome editing in mouse embryos using recombinant adeno-associated viruses
    Article Snippet: .. Purified PCR product was cloned into the pCRTM -Blunt II-TOPO vector using Zero Blunt TOPO PCR Cloning Kit (Thermo Fisher Sci. .. Cat. No. K280002) and sequenced using the Sanger method.

    Polymerase Chain Reaction:

    Article Title: Streamlined ex vivo and in vivo genome editing in mouse embryos using recombinant adeno-associated viruses
    Article Snippet: .. Purified PCR products were cloned into the pCRTM -Blunt II-TOPO vector using Zero Blunt TOPO PCR Cloning Kit (Thermo Fisher Sci. .. Cat. No. K280002), and used to transform DH5α Escherichia coli bacteria.

    Article Title: Streamlined ex vivo and in vivo genome editing in mouse embryos using recombinant adeno-associated viruses
    Article Snippet: .. Purified PCR product was cloned into the pCRTM -Blunt II-TOPO vector using Zero Blunt TOPO PCR Cloning Kit (Thermo Fisher Sci. .. Cat. No. K280002) and sequenced using the Sanger method.

    Article Title: Elimination of 01/A′–A0 pre-rRNA processing by-product in human cells involves cooperative action of two nuclear exosome-associated nucleases: RRP6 and DIS3
    Article Snippet: .. The final PCR products were cloned with the use of Zero Blunt Topo PCR Cloning Kit (Invitrogen), and the inserts were sequenced using M13 Forward (−20) and M13 Reverse oligonucleotides, following recommendations of the manufacturer. .. High-throughput analysis of the RNA 3′-ends was performed essentially as described previously , with some modifications listed hereafter, using 2 µg of total RNA as a starting material.

    Article Title: CRISPR/Cas9 Beta-globin Gene Targeting in Human Hematopoietic Stem Cells
    Article Snippet: .. The PCR product was gel-purified and re-amplified using a nested primer set ( HBB _nested_fw: 5’-GAAGATATGCTTAGAACCGAGG-3’ and HBB _nested_rw: 5’-CCACATGCCCAGTTTCTATTGG-3’) to create a 685bp PCR amplicon (see ) that was gel-purified and cloned into a TOPO plasmid using the Zero Blunt TOPO PCR Cloning Kit (Life Technologies) according to the manufacturer's protocol. .. TOPO reactions were transformed into XL-1 Blue competent cells, plated on kanamycin-containing agar plates, and single colonies were sequenced by McLab (South San Francisco, CA, USA) by rolling circle amplification followed by sequencing using the following primer: 5′-GAAGATATGCTTAGAACCGAGG-3′.

    Staining:

    Article Title: Evaluation of Lassa virus vaccine immunogenicity in a CBA/J-ML29 mouse model
    Article Snippet: .. Cells were washed with PBS and surface staining was performed using the following markers: CD3 (eBioscience, Cat. no. 45-0031, clone 145-2C11, PerCP-Cy5.5) and CD4 (eBioscience, Cat. no. 17-0042, clone RM4-5, APC) or CD3 and CD8 (eBioscience, Cat. no. 17-0081, clone 53-6.7, APC). .. Cells were then washed with PBS, fixed/permeabilized using Cytofix/Cytoperm™ Plus kit (BD Bioscience, Cat. no. 555028) and stained for IFN-γ (eBioscience, Cat. no. 12-7311, clone XMG1.2, PE) and TNF-α (eBioscience, Cat. no. 11-7321, clone MP6-XT22, FITC).

    Plasmid Preparation:

    Article Title: Streamlined ex vivo and in vivo genome editing in mouse embryos using recombinant adeno-associated viruses
    Article Snippet: .. Purified PCR products were cloned into the pCRTM -Blunt II-TOPO vector using Zero Blunt TOPO PCR Cloning Kit (Thermo Fisher Sci. .. Cat. No. K280002), and used to transform DH5α Escherichia coli bacteria.

    Article Title: Streamlined ex vivo and in vivo genome editing in mouse embryos using recombinant adeno-associated viruses
    Article Snippet: .. Purified PCR product was cloned into the pCRTM -Blunt II-TOPO vector using Zero Blunt TOPO PCR Cloning Kit (Thermo Fisher Sci. .. Cat. No. K280002) and sequenced using the Sanger method.

    Article Title: CRISPR/Cas9 Beta-globin Gene Targeting in Human Hematopoietic Stem Cells
    Article Snippet: .. The PCR product was gel-purified and re-amplified using a nested primer set ( HBB _nested_fw: 5’-GAAGATATGCTTAGAACCGAGG-3’ and HBB _nested_rw: 5’-CCACATGCCCAGTTTCTATTGG-3’) to create a 685bp PCR amplicon (see ) that was gel-purified and cloned into a TOPO plasmid using the Zero Blunt TOPO PCR Cloning Kit (Life Technologies) according to the manufacturer's protocol. .. TOPO reactions were transformed into XL-1 Blue competent cells, plated on kanamycin-containing agar plates, and single colonies were sequenced by McLab (South San Francisco, CA, USA) by rolling circle amplification followed by sequencing using the following primer: 5′-GAAGATATGCTTAGAACCGAGG-3′.

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    Thermo Fisher top10 escherichia coli
    DNA methylation in the enhancer region regulates perforin transcription. (a) Promoter region sequence containing enhancer in the PRF1 gene was aligned between human and mouse using the VISTA tool. The conservation degree is indicated by the peaks, with coloured peaks (blue for exon and light red for the non‐coding sequence) indicating > 70% conservation. Ten cytosine–phosphate–guanine sites (CpGs) in the enhancer are displayed upstream from the transcription start site (TSS). (b) Sorted CD8 + T cells from healthy donors were cultured initially in the presence of 200 IU/ml recombinant human interleukin (IL)‐2, 5 μg/ml plate‐coated anti‐CD3 and 1 μg/ml anti‐CD28 stimulating antibodies in vitro . 5‐Azacytidine (5‐aza) was added to the culture at a final concentration of 2·5 μM/ml for 48 h, followed by culture medium replacement and incubation for another 48 h. Flow cytometry data demonstrating perforin expression pre‐ and post‐treatment are shown. (c) Perforin – and perforin + CD8 + T cells were sorted. DNA was extracted and bisulfite‐converted from each cell subset, TA‐cloned to pCR4‐TOPO vector and transformed into <t>TOP10</t> <t>Escherichia</t> coli . Colonies were picked from each cell subset and their DNA was sequenced to measure DNA methylation status of the 10 CpGs in the enhancer. The data display rows represent individually sequenced clone. (d) The bar graphs display total DNA methylation percentage in each CpG from clones in (c).
    Top10 Escherichia Coli, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher e coli top10 carrying vector pcr4 topo
    Resistance against sulfonamide antibiotics mediated by SEW2_dhps01, SEW5_dhps01, AEW9_dhps01 , and AEG2_dhps01 . Five microliters of serially diluted E. coli <t>TOP10</t> cultures with starting OD 600 of 0.5 were spotted onto Iso-Sensitest agar plates supplemented with 1000 mg/L sulfamethazine (+ SMZ), 250 mg/L sulfamethoxazole (+ SMX), 250 mg/L sulfadiazine (+ SDZ) or 500 mg/L sulfisoxazole (+ SOX). Iso-Sensitest agar plates with no sulfonamide added (control) were also included. E. coli TOP10 cultures carrying the cloning vector <t>pCR4-TOPO,</t> pCR4_SEW2_dhps01, pCR4_SEW5_dhps01, pCR4_AEW9_dhps01 or pCR4_AEG2_dhps01 were considered.
    E Coli Top10 Carrying Vector Pcr4 Topo, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher escherichia coli
    Representative SDS-PAGE, 2-dimensional electrophoresis (2DE), and Western blots showing the workflow for selection of immunoreactive spots. (A) The 1-dimensional SDS-PAGE image of Staphylococcus aureus Newbould 305 trypsinized protein (lane 1) and <t>Escherichia</t> coli trypsinized protein (lane 2). (B) 2DE gel conducted on 7 cm, pH 4 to 7, immobilized pH gradient (IPG) strips of S. aureus Newbould 305 trypsinized protein. Mr = molecular weight. (C) Immunoblot of S. aureus Newbould 305 trypsinized protein using pooled bovine mastitic milk, with immunoreactive regions highlighted in red circles. (D) 2DE gel conducted on 7 cm, pH 4 to 7, IPG strips of Escherichia coli DH10-β trypsinized protein. (E) Immunoblot of E. coli DH10-β trypsinized protein using bovine mastitic milk, with the immunoreactive regions highlighted in red circles. Color version available online.
    Escherichia Coli, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 76 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    Thermo Fisher multishot flexplate top10 competent cells
    Analysis of the pop transcriptional unit. (A) Promoter activity assay for the promoter of pop , which was introduced in the promoterless GFP vector pProbe-NT. Mean fluorescence units of E. coli <t>Top10</t> cells with the different constructs in culture conditions as indicated are given. Error bars show standard deviations. Statistical significance between empty vector (grey bars) and the promoter construct (blue bars) and between growth conditions was tested with a Welch two sample t-test (**/++ p≤0.01; *** p≤0.001; ns, not significant). (B) Test for mono- or polycistronic mRNA. An agarose gel of RT-PCRs is shown. Two different forward primers, binding within ycbG (no. 23) or within pop (no. 24), were combined with a pop reverse primer (no. 25). L: 100 bp DNA Ladder (NEB); 23: PCR with primers 23+25; 24: PCR with primers 24+25. (C) Test for the predicted rho-independent terminator. An agarose gel of RT-PCRs is shown. Two different reverse primers, binding upstream (no. 27) or downstream (no. 28) of the stem loop structure, were combined with a pop forward primer (no. 26). L, 1 kb DNA Ladder (NEB); 27, PCR with primers 27+26; 28, PCR with primers 28+26; d. ORFs, downstream ORFs.
    Multishot Flexplate Top10 Competent Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    DNA methylation in the enhancer region regulates perforin transcription. (a) Promoter region sequence containing enhancer in the PRF1 gene was aligned between human and mouse using the VISTA tool. The conservation degree is indicated by the peaks, with coloured peaks (blue for exon and light red for the non‐coding sequence) indicating > 70% conservation. Ten cytosine–phosphate–guanine sites (CpGs) in the enhancer are displayed upstream from the transcription start site (TSS). (b) Sorted CD8 + T cells from healthy donors were cultured initially in the presence of 200 IU/ml recombinant human interleukin (IL)‐2, 5 μg/ml plate‐coated anti‐CD3 and 1 μg/ml anti‐CD28 stimulating antibodies in vitro . 5‐Azacytidine (5‐aza) was added to the culture at a final concentration of 2·5 μM/ml for 48 h, followed by culture medium replacement and incubation for another 48 h. Flow cytometry data demonstrating perforin expression pre‐ and post‐treatment are shown. (c) Perforin – and perforin + CD8 + T cells were sorted. DNA was extracted and bisulfite‐converted from each cell subset, TA‐cloned to pCR4‐TOPO vector and transformed into TOP10 Escherichia coli . Colonies were picked from each cell subset and their DNA was sequenced to measure DNA methylation status of the 10 CpGs in the enhancer. The data display rows represent individually sequenced clone. (d) The bar graphs display total DNA methylation percentage in each CpG from clones in (c).

    Journal: Clinical and Experimental Immunology

    Article Title: Tissue‐resident memory T cells are epigenetically cytotoxic with signs of exhaustion in human urinary bladder cancer

    doi: 10.1111/cei.13183

    Figure Lengend Snippet: DNA methylation in the enhancer region regulates perforin transcription. (a) Promoter region sequence containing enhancer in the PRF1 gene was aligned between human and mouse using the VISTA tool. The conservation degree is indicated by the peaks, with coloured peaks (blue for exon and light red for the non‐coding sequence) indicating > 70% conservation. Ten cytosine–phosphate–guanine sites (CpGs) in the enhancer are displayed upstream from the transcription start site (TSS). (b) Sorted CD8 + T cells from healthy donors were cultured initially in the presence of 200 IU/ml recombinant human interleukin (IL)‐2, 5 μg/ml plate‐coated anti‐CD3 and 1 μg/ml anti‐CD28 stimulating antibodies in vitro . 5‐Azacytidine (5‐aza) was added to the culture at a final concentration of 2·5 μM/ml for 48 h, followed by culture medium replacement and incubation for another 48 h. Flow cytometry data demonstrating perforin expression pre‐ and post‐treatment are shown. (c) Perforin – and perforin + CD8 + T cells were sorted. DNA was extracted and bisulfite‐converted from each cell subset, TA‐cloned to pCR4‐TOPO vector and transformed into TOP10 Escherichia coli . Colonies were picked from each cell subset and their DNA was sequenced to measure DNA methylation status of the 10 CpGs in the enhancer. The data display rows represent individually sequenced clone. (d) The bar graphs display total DNA methylation percentage in each CpG from clones in (c).

    Article Snippet: Next, the PCR amplicons were TA‐cloned into pCR4‐TOPO vector (ThermoFisher) and transformed into TOP10 Escherichia coli (ThermoFisher) by heat shock, according to the manufacturer’s protocol.

    Techniques: DNA Methylation Assay, Sequencing, Cell Culture, Recombinant, In Vitro, Concentration Assay, Incubation, Flow Cytometry, Cytometry, Expressing, Clone Assay, Plasmid Preparation, Transformation Assay

    Resistance against sulfonamide antibiotics mediated by SEW2_dhps01, SEW5_dhps01, AEW9_dhps01 , and AEG2_dhps01 . Five microliters of serially diluted E. coli TOP10 cultures with starting OD 600 of 0.5 were spotted onto Iso-Sensitest agar plates supplemented with 1000 mg/L sulfamethazine (+ SMZ), 250 mg/L sulfamethoxazole (+ SMX), 250 mg/L sulfadiazine (+ SDZ) or 500 mg/L sulfisoxazole (+ SOX). Iso-Sensitest agar plates with no sulfonamide added (control) were also included. E. coli TOP10 cultures carrying the cloning vector pCR4-TOPO, pCR4_SEW2_dhps01, pCR4_SEW5_dhps01, pCR4_AEW9_dhps01 or pCR4_AEG2_dhps01 were considered.

    Journal: Frontiers in Microbiology

    Article Title: Discovery of Novel Antibiotic Resistance Determinants in Forest and Grassland Soil Metagenomes

    doi: 10.3389/fmicb.2019.00460

    Figure Lengend Snippet: Resistance against sulfonamide antibiotics mediated by SEW2_dhps01, SEW5_dhps01, AEW9_dhps01 , and AEG2_dhps01 . Five microliters of serially diluted E. coli TOP10 cultures with starting OD 600 of 0.5 were spotted onto Iso-Sensitest agar plates supplemented with 1000 mg/L sulfamethazine (+ SMZ), 250 mg/L sulfamethoxazole (+ SMX), 250 mg/L sulfadiazine (+ SDZ) or 500 mg/L sulfisoxazole (+ SOX). Iso-Sensitest agar plates with no sulfonamide added (control) were also included. E. coli TOP10 cultures carrying the cloning vector pCR4-TOPO, pCR4_SEW2_dhps01, pCR4_SEW5_dhps01, pCR4_AEW9_dhps01 or pCR4_AEG2_dhps01 were considered.

    Article Snippet: E. coli TOP10 carrying vector pCR4-TOPO (Thermo Fisher Scientific) was used as control.

    Techniques: Clone Assay, Plasmid Preparation

    Antibiotic susceptibility profiles of E. coli TOP10 carrying soil-derived genes involved in antibiotic resistance. The genes were subcloned into plasmid vector pCR4-TOPO. MICs of antibiotics were determined using the broth microdilution method and are presented as fold increase relative to those for E. coli TOP10 carrying the cloning vector pCR4-TOPO. CAX, cefotaxime; CHL, chloramphenicol; ERY, erythromycin; GEN, gentamicin; LIN, lincomycin; RIF, rifampicin; SDZ, sulfadiazine; SMX, sulfamethoxazole; SMZ, sulfamethazine; SOX, sulfisoxazole; TET, tetracycline; TYL, tylosin.

    Journal: Frontiers in Microbiology

    Article Title: Discovery of Novel Antibiotic Resistance Determinants in Forest and Grassland Soil Metagenomes

    doi: 10.3389/fmicb.2019.00460

    Figure Lengend Snippet: Antibiotic susceptibility profiles of E. coli TOP10 carrying soil-derived genes involved in antibiotic resistance. The genes were subcloned into plasmid vector pCR4-TOPO. MICs of antibiotics were determined using the broth microdilution method and are presented as fold increase relative to those for E. coli TOP10 carrying the cloning vector pCR4-TOPO. CAX, cefotaxime; CHL, chloramphenicol; ERY, erythromycin; GEN, gentamicin; LIN, lincomycin; RIF, rifampicin; SDZ, sulfadiazine; SMX, sulfamethoxazole; SMZ, sulfamethazine; SOX, sulfisoxazole; TET, tetracycline; TYL, tylosin.

    Article Snippet: E. coli TOP10 carrying vector pCR4-TOPO (Thermo Fisher Scientific) was used as control.

    Techniques: Derivative Assay, Plasmid Preparation, Clone Assay

    Representative SDS-PAGE, 2-dimensional electrophoresis (2DE), and Western blots showing the workflow for selection of immunoreactive spots. (A) The 1-dimensional SDS-PAGE image of Staphylococcus aureus Newbould 305 trypsinized protein (lane 1) and Escherichia coli trypsinized protein (lane 2). (B) 2DE gel conducted on 7 cm, pH 4 to 7, immobilized pH gradient (IPG) strips of S. aureus Newbould 305 trypsinized protein. Mr = molecular weight. (C) Immunoblot of S. aureus Newbould 305 trypsinized protein using pooled bovine mastitic milk, with immunoreactive regions highlighted in red circles. (D) 2DE gel conducted on 7 cm, pH 4 to 7, IPG strips of Escherichia coli DH10-β trypsinized protein. (E) Immunoblot of E. coli DH10-β trypsinized protein using bovine mastitic milk, with the immunoreactive regions highlighted in red circles. Color version available online.

    Journal: Journal of dairy science

    Article Title: Immunoproteomics to identify Staphylococcus aureus antigens expressed in bovine milk during mastitis

    doi: 10.3168/jds.2017-14040

    Figure Lengend Snippet: Representative SDS-PAGE, 2-dimensional electrophoresis (2DE), and Western blots showing the workflow for selection of immunoreactive spots. (A) The 1-dimensional SDS-PAGE image of Staphylococcus aureus Newbould 305 trypsinized protein (lane 1) and Escherichia coli trypsinized protein (lane 2). (B) 2DE gel conducted on 7 cm, pH 4 to 7, immobilized pH gradient (IPG) strips of S. aureus Newbould 305 trypsinized protein. Mr = molecular weight. (C) Immunoblot of S. aureus Newbould 305 trypsinized protein using pooled bovine mastitic milk, with immunoreactive regions highlighted in red circles. (D) 2DE gel conducted on 7 cm, pH 4 to 7, IPG strips of Escherichia coli DH10-β trypsinized protein. (E) Immunoblot of E. coli DH10-β trypsinized protein using bovine mastitic milk, with the immunoreactive regions highlighted in red circles. Color version available online.

    Article Snippet: Briefly, S. aureus Newbould 305, S. aureus C1, and Escherichia coli (DH10-β Top10, Thermo Fisher Scientific, Waltham, MA) were grown in LIM overnight to an optical density ( OD ) of 0.75 to 1.2 and harvested by centrifugation (6,000 × g for 10 min at 4°C), before washing 3 times with 1× PBS.

    Techniques: SDS Page, Electrophoresis, Two-Dimensional Gel Electrophoresis, Western Blot, Selection, Molecular Weight

    Analysis of the pop transcriptional unit. (A) Promoter activity assay for the promoter of pop , which was introduced in the promoterless GFP vector pProbe-NT. Mean fluorescence units of E. coli Top10 cells with the different constructs in culture conditions as indicated are given. Error bars show standard deviations. Statistical significance between empty vector (grey bars) and the promoter construct (blue bars) and between growth conditions was tested with a Welch two sample t-test (**/++ p≤0.01; *** p≤0.001; ns, not significant). (B) Test for mono- or polycistronic mRNA. An agarose gel of RT-PCRs is shown. Two different forward primers, binding within ycbG (no. 23) or within pop (no. 24), were combined with a pop reverse primer (no. 25). L: 100 bp DNA Ladder (NEB); 23: PCR with primers 23+25; 24: PCR with primers 24+25. (C) Test for the predicted rho-independent terminator. An agarose gel of RT-PCRs is shown. Two different reverse primers, binding upstream (no. 27) or downstream (no. 28) of the stem loop structure, were combined with a pop forward primer (no. 26). L, 1 kb DNA Ladder (NEB); 27, PCR with primers 27+26; 28, PCR with primers 28+26; d. ORFs, downstream ORFs.

    Journal: bioRxiv

    Article Title: A novel pH-regulated, unusual 603 bp overlapping protein coding gene pop is encoded antisense to ompA in Escherichia coli O157:H7 (EHEC)

    doi: 10.1101/852251

    Figure Lengend Snippet: Analysis of the pop transcriptional unit. (A) Promoter activity assay for the promoter of pop , which was introduced in the promoterless GFP vector pProbe-NT. Mean fluorescence units of E. coli Top10 cells with the different constructs in culture conditions as indicated are given. Error bars show standard deviations. Statistical significance between empty vector (grey bars) and the promoter construct (blue bars) and between growth conditions was tested with a Welch two sample t-test (**/++ p≤0.01; *** p≤0.001; ns, not significant). (B) Test for mono- or polycistronic mRNA. An agarose gel of RT-PCRs is shown. Two different forward primers, binding within ycbG (no. 23) or within pop (no. 24), were combined with a pop reverse primer (no. 25). L: 100 bp DNA Ladder (NEB); 23: PCR with primers 23+25; 24: PCR with primers 24+25. (C) Test for the predicted rho-independent terminator. An agarose gel of RT-PCRs is shown. Two different reverse primers, binding upstream (no. 27) or downstream (no. 28) of the stem loop structure, were combined with a pop forward primer (no. 26). L, 1 kb DNA Ladder (NEB); 27, PCR with primers 27+26; 28, PCR with primers 28+26; d. ORFs, downstream ORFs.

    Article Snippet: Vector constructs were transformed in E. coli Top10 cells and plated on LB with required antibiotics.

    Techniques: Activity Assay, Plasmid Preparation, Fluorescence, Construct, Agarose Gel Electrophoresis, Binding Assay, Polymerase Chain Reaction