att b2 1 sites  (Thermo Fisher)


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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.
    Att B2 1 Sites, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 19517 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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 "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

    5) 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

    6) 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

    7) 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

    8) Product Images from "Allelic differences of clustered terpene synthases contribute to correlated intra-specific variation of floral and herbivory-induced volatiles in a wild tobacco"

    Article Title: Allelic differences of clustered terpene synthases contribute to correlated intra-specific variation of floral and herbivory-induced volatiles in a wild tobacco

    Journal: bioRxiv

    doi: 10.1101/2020.04.26.062133

    Allelic expression of NaTPS38 in F1 hybrids of Arizona and Utah genotypes. ( a ) Scheme of primers used to measure the allele-specific transcript abundance. Green arrows refer to primers that can specifically amplify the Utah allele and blue arrows refer to primers that can specifically amplify the Arizona allele. Primers (grey arrows) that do not distinguish the two alleles were also used to measure total transcript abundance of NaTPS38 . ( b ) Expression differences of NaTPS38 alleles between F0 and F1 plants. Plants were treated with wounding plus oral secretion (N=5). Mean and SE are shown. Student’s t -test was used to determine differences between F0 and F1 plants. ns refers to no significant difference was found. ( c ) Expression differences of alleles in F1 individuals. Plants were treated with wounding plus oral secretion (N=5). Each dot represents an individual plant. Difference between two alleles was determined by paired Student’s t -test. Blue and green colors indicate alleles from Arizona and Utah genotypes, respectively.
    Figure Legend Snippet: Allelic expression of NaTPS38 in F1 hybrids of Arizona and Utah genotypes. ( a ) Scheme of primers used to measure the allele-specific transcript abundance. Green arrows refer to primers that can specifically amplify the Utah allele and blue arrows refer to primers that can specifically amplify the Arizona allele. Primers (grey arrows) that do not distinguish the two alleles were also used to measure total transcript abundance of NaTPS38 . ( b ) Expression differences of NaTPS38 alleles between F0 and F1 plants. Plants were treated with wounding plus oral secretion (N=5). Mean and SE are shown. Student’s t -test was used to determine differences between F0 and F1 plants. ns refers to no significant difference was found. ( c ) Expression differences of alleles in F1 individuals. Plants were treated with wounding plus oral secretion (N=5). Each dot represents an individual plant. Difference between two alleles was determined by paired Student’s t -test. Blue and green colors indicate alleles from Arizona and Utah genotypes, respectively.

    Techniques Used: Expressing

    Allelic variations of the terpene cluster are associated with differences in floral volatiles and HIPVs among N. attenuata natural accessions. ( a ) Three different haplotypes found from the open reading frame of NaTPS38 from N. attenuata accessions. The amino acid sequence of NaTPS38 from the Utah genotype is set as the reference, and the different haplotype loci are marked in red. The presence of intact NaTPS25 gene in the natural accessions are shown on the right (+, present; -not present). ( b ) The emission of ( E )-α-bergamotene and ( E )-β-ocimene in both flowers and herbivory-induced leaves are different among three NaTPS38 haplotypes. The data for ( E )-α-bergamotene emission were extract from our previous study ( Zhou et al ., 2017 ). Mean and SE are shown. Statistical differences among haplotypes are determined by ANOVA ( P
    Figure Legend Snippet: Allelic variations of the terpene cluster are associated with differences in floral volatiles and HIPVs among N. attenuata natural accessions. ( a ) Three different haplotypes found from the open reading frame of NaTPS38 from N. attenuata accessions. The amino acid sequence of NaTPS38 from the Utah genotype is set as the reference, and the different haplotype loci are marked in red. The presence of intact NaTPS25 gene in the natural accessions are shown on the right (+, present; -not present). ( b ) The emission of ( E )-α-bergamotene and ( E )-β-ocimene in both flowers and herbivory-induced leaves are different among three NaTPS38 haplotypes. The data for ( E )-α-bergamotene emission were extract from our previous study ( Zhou et al ., 2017 ). Mean and SE are shown. Statistical differences among haplotypes are determined by ANOVA ( P

    Techniques Used: Sequencing

    9) 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

    10) 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

    11) 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

    12) 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

    13) 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

    14) 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

    15) 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

    16) 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

    17) 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

    18) 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

    19) Product Images from "OXA-198, an Acquired Carbapenem-Hydrolyzing Class D ?-Lactamase from Pseudomonas aeruginosa ▿"

    Article Title: OXA-198, an Acquired Carbapenem-Hydrolyzing Class D ?-Lactamase from Pseudomonas aeruginosa ▿

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.00522-11

    Schematic representation of the genetic environment of the bla OXA-198 gene. The 5′ conserved segment (5′-CS) contains the integrase gene intI1 , while the 3′-CS contains the qacE Δ 1 , sul1 , and orf5 genes typical of class I integrons. Filled circles indicate attC sites. Arrows indicate the direction of transcription of the coding regions. IRi of the Tn 6060 -like sequence is shown. paeR7IM and paeR7IN code for an adenine-specific methyltransferase and a DNA invertase, respectively. Genes tniB and tniA , involved in transposition, are located downstream of orf5 .
    Figure Legend Snippet: Schematic representation of the genetic environment of the bla OXA-198 gene. The 5′ conserved segment (5′-CS) contains the integrase gene intI1 , while the 3′-CS contains the qacE Δ 1 , sul1 , and orf5 genes typical of class I integrons. Filled circles indicate attC sites. Arrows indicate the direction of transcription of the coding regions. IRi of the Tn 6060 -like sequence is shown. paeR7IM and paeR7IN code for an adenine-specific methyltransferase and a DNA invertase, respectively. Genes tniB and tniA , involved in transposition, are located downstream of orf5 .

    Techniques Used: Sequencing

    Microbiological assay plate showing inactivation of meropenem (A) (central disk) or imipenem (B) by the OXA-198 enzyme. MH agar plates were inoculated with the carbapenem-susceptible E. coli ATCC25922 strain. A central disk of meropenem (10 μg) or imipenem (10 μg) was put on the dish with 4 satellite disks containing 20 μl of crude sonicated extract of E. coli TOP10 expressing OXA-198 (nitrocefin-specific activity, 1.05 μmol/min/mg) (1), 10 μl of extract (2), 5 μl of extract (3), and 20 μl of phosphate-buffered saline (4). MEM, meropenem; IPM, imipenem.
    Figure Legend Snippet: Microbiological assay plate showing inactivation of meropenem (A) (central disk) or imipenem (B) by the OXA-198 enzyme. MH agar plates were inoculated with the carbapenem-susceptible E. coli ATCC25922 strain. A central disk of meropenem (10 μg) or imipenem (10 μg) was put on the dish with 4 satellite disks containing 20 μl of crude sonicated extract of E. coli TOP10 expressing OXA-198 (nitrocefin-specific activity, 1.05 μmol/min/mg) (1), 10 μl of extract (2), 5 μl of extract (3), and 20 μl of phosphate-buffered saline (4). MEM, meropenem; IPM, imipenem.

    Techniques Used: Microbial Assay, Sonication, Expressing, Activity Assay

    20) Product Images from "Complexity of Bidirectional Transcription and Alternative Splicing at Human RCAN3 Locus"

    Article Title: Complexity of Bidirectional Transcription and Alternative Splicing at Human RCAN3 Locus

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0024508

    RCAN3AS mRNA isoforms sequence identification and analysis. A) 1% agarose gel loaded with RCAN3AS colony PCR products. From left to right: RCAN3AS-1,3 (expected band size: 547 bp), RCAN3AS-1,2a,3 (expected band size: 658 bp), RCAN3AS-1,2b,3 (expected band size: 699 bp) and RCAN3AS-1,2,3 (expected band size: 606 bp). M: size marker GeneRuler (500 ng). Band heights result by using specific pCR2.1 plasmid primer pairs. B) RCAN3AS mRNA isoform sequences aligned by ClustalW software. Exon 2a and exon 2b are longer than exon 2 in their 5′ (52 bp and 93 bp, respectively).
    Figure Legend Snippet: RCAN3AS mRNA isoforms sequence identification and analysis. A) 1% agarose gel loaded with RCAN3AS colony PCR products. From left to right: RCAN3AS-1,3 (expected band size: 547 bp), RCAN3AS-1,2a,3 (expected band size: 658 bp), RCAN3AS-1,2b,3 (expected band size: 699 bp) and RCAN3AS-1,2,3 (expected band size: 606 bp). M: size marker GeneRuler (500 ng). Band heights result by using specific pCR2.1 plasmid primer pairs. B) RCAN3AS mRNA isoform sequences aligned by ClustalW software. Exon 2a and exon 2b are longer than exon 2 in their 5′ (52 bp and 93 bp, respectively).

    Techniques Used: Sequencing, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Marker, Plasmid Preparation, Software

    21) Product Images from "FK-16 Derived from the Anticancer Peptide LL-37 Induces Caspase-Independent Apoptosis and Autophagic Cell Death in Colon Cancer Cells"

    Article Title: FK-16 Derived from the Anticancer Peptide LL-37 Induces Caspase-Independent Apoptosis and Autophagic Cell Death in Colon Cancer Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0063641

    Schematic diagram showing the common regulation of caspase-independent apoptosis and autophagic cell death by the p53-Bcl-2/Bax cascade and their reciprocal regulation induced by FK-16 in colon cancer cells.
    Figure Legend Snippet: Schematic diagram showing the common regulation of caspase-independent apoptosis and autophagic cell death by the p53-Bcl-2/Bax cascade and their reciprocal regulation induced by FK-16 in colon cancer cells.

    Techniques Used:

    Activation of p53 was required for AIF/EndoG-dependent apoptosis and autophagic cell death induced by FK-16. ( A ) HCT116 cells were incubated with FK-16 for 24 h or 48 h. Cytosolic and nuclear p53 and total expression of Bcl-2 members (PUMA, Bcl-2, Bax and Bak) were determined by Western blot. GAPDH and Lamin A/C were used as internal controls for cytosolic and nuclear proteins, respectively. ( B ) Knockdown of p53 reversed the upregulation of pro-autophagic factors (Atg5 and Atg7) and pro-apoptotic factors (Bax, Bak, nuclear AIF and nuclear EndoG) as well as downregulation of Bcl-2 by FK-16. ( C ) FK-16 failed to induced phosphotidylserine externalization in p53-depleted HCT116 cells. After transfection with control- or p53-siRNA for 48 h, cells were treated with or without FK-16 (40 µM) for another 24 h followed by propidium iodide/annexin V-double staining. ( D ) Knockdown of 53 markedly reduced the number of LC3 + autophagic vacuoles in FK-16-treated cells (40 µM; 48 h) as determined by confocal immunofluorescence (400×). Nuclei (blue) were stained with DAPI. ( E ) Knockdown of p53 partially reversed the inhibitory effect of FK-16 on cell viability in HCT116 as determined by MTT assay. Data are presented as means ± S.D. of three separate experiments. *, p
    Figure Legend Snippet: Activation of p53 was required for AIF/EndoG-dependent apoptosis and autophagic cell death induced by FK-16. ( A ) HCT116 cells were incubated with FK-16 for 24 h or 48 h. Cytosolic and nuclear p53 and total expression of Bcl-2 members (PUMA, Bcl-2, Bax and Bak) were determined by Western blot. GAPDH and Lamin A/C were used as internal controls for cytosolic and nuclear proteins, respectively. ( B ) Knockdown of p53 reversed the upregulation of pro-autophagic factors (Atg5 and Atg7) and pro-apoptotic factors (Bax, Bak, nuclear AIF and nuclear EndoG) as well as downregulation of Bcl-2 by FK-16. ( C ) FK-16 failed to induced phosphotidylserine externalization in p53-depleted HCT116 cells. After transfection with control- or p53-siRNA for 48 h, cells were treated with or without FK-16 (40 µM) for another 24 h followed by propidium iodide/annexin V-double staining. ( D ) Knockdown of 53 markedly reduced the number of LC3 + autophagic vacuoles in FK-16-treated cells (40 µM; 48 h) as determined by confocal immunofluorescence (400×). Nuclei (blue) were stained with DAPI. ( E ) Knockdown of p53 partially reversed the inhibitory effect of FK-16 on cell viability in HCT116 as determined by MTT assay. Data are presented as means ± S.D. of three separate experiments. *, p

    Techniques Used: Activation Assay, Incubation, Expressing, Western Blot, Transfection, Double Staining, Immunofluorescence, Staining, MTT Assay

    Altered expression of Bcl-2 and Bax was required for AIF/EndoG-dependent apoptosis and autophagic cell death induced by FK-16. ( A ) Restoration of Bcl-2 expression by transfection with Bcl-2-encoding plasmid reversed the upregulation of pro-autophagic (Atg5, Atg7 and LC3-I/II) and pro-apoptotic (nuclear AIF and nuclear EndoG) factors induced by FK-16 (40 µM; 48 h) in HCT116. ( B ) Genetic ablation of Bax (Bax KO) in HCT116 reversed FK-16-induced apoptotic and autophagic signals. Bax +/− HCT116 treated with or without FK-16 was used as control. ( C ) Restoration of Bcl-2 expression or genetic ablation of Bax in HCT116 abolished the formation of LC3 + autophagic vacuoles induced by FK-16 (40 µM; 48 h) as determined by confocal immunofluorescence (400×). ( D ) Restoration of Bcl-2 expression or genetic ablation of Bax partially reversed the inhibitory effect of FK-16 on cell viability in HCT116 as determined by MTT assay. Data are presented as means ± S.D. of three separate experiments. *, p
    Figure Legend Snippet: Altered expression of Bcl-2 and Bax was required for AIF/EndoG-dependent apoptosis and autophagic cell death induced by FK-16. ( A ) Restoration of Bcl-2 expression by transfection with Bcl-2-encoding plasmid reversed the upregulation of pro-autophagic (Atg5, Atg7 and LC3-I/II) and pro-apoptotic (nuclear AIF and nuclear EndoG) factors induced by FK-16 (40 µM; 48 h) in HCT116. ( B ) Genetic ablation of Bax (Bax KO) in HCT116 reversed FK-16-induced apoptotic and autophagic signals. Bax +/− HCT116 treated with or without FK-16 was used as control. ( C ) Restoration of Bcl-2 expression or genetic ablation of Bax in HCT116 abolished the formation of LC3 + autophagic vacuoles induced by FK-16 (40 µM; 48 h) as determined by confocal immunofluorescence (400×). ( D ) Restoration of Bcl-2 expression or genetic ablation of Bax partially reversed the inhibitory effect of FK-16 on cell viability in HCT116 as determined by MTT assay. Data are presented as means ± S.D. of three separate experiments. *, p

    Techniques Used: Expressing, Transfection, Plasmid Preparation, Immunofluorescence, MTT Assay

    22) Product Images from "Phagocytosis-like cell engulfment by a planctomycete bacterium"

    Article Title: Phagocytosis-like cell engulfment by a planctomycete bacterium

    Journal: Nature Communications

    doi: 10.1038/s41467-019-13499-2

    Light and fluorescent micrographs of ‘ Candidatus Uab amorphum’. a Cells of ‘ Ca . Uab amorphum’ (arrowheads) in the xenic culture. b , c A cell of ‘ Ca . Uab amorphum’. Double arrowheads indicate an engulfed bacterium. d – i Selected images of time-lapse video showing prey engulfment process of ‘ Ca . Uab amorphum’. j – o Selected images of confocal fluorescent time-lapse video showing prey engulfment and digestion process of ‘ Ca . Uab amorphum’. Green fluorescence indicates AcGFP1-labelled Escherichia coli . Scale bars, 10 μm ( a ) and 5 μm ( b – o ).
    Figure Legend Snippet: Light and fluorescent micrographs of ‘ Candidatus Uab amorphum’. a Cells of ‘ Ca . Uab amorphum’ (arrowheads) in the xenic culture. b , c A cell of ‘ Ca . Uab amorphum’. Double arrowheads indicate an engulfed bacterium. d – i Selected images of time-lapse video showing prey engulfment process of ‘ Ca . Uab amorphum’. j – o Selected images of confocal fluorescent time-lapse video showing prey engulfment and digestion process of ‘ Ca . Uab amorphum’. Green fluorescence indicates AcGFP1-labelled Escherichia coli . Scale bars, 10 μm ( a ) and 5 μm ( b – o ).

    Techniques Used: Fluorescence

    23) 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

    24) Product Images from "Evolution of herbivore-induced early defense signaling was shaped by genome-wide duplications in Nicotiana"

    Article Title: Evolution of herbivore-induced early defense signaling was shaped by genome-wide duplications in Nicotiana

    Journal: eLife

    doi: 10.7554/eLife.19531

    In N. attenuata , OS Ms induced higher NaLRRK1 transcript levels in 35S- jmt /ir- mje plants than in WT plants. ( a ) diagram shows the JA metabolic flux in WT (left) and 35S- jmt /ir- mje plants (right). ( b ) in comparison to WT plants, 35S- jmt /ir- mje plants have reduced level of OS Ms -induced JA-Ile. ( c ) the transcript levels of NaLRRK1 are higher in 35S- jmt /ir- mje plants than in WT plants. In both panel b and c, filled and unfilled bars indicate 35S jmt /ir- mje and WT plants, respectively. Data are presented as means ± SEM. Rosette stage N. attenuata plants were wounded and 20 µL of OS Ms was immediately applied to the wounds. Leaf samples were collected at 1 hr after elicitations. Asterisk indicates significant differences between WT and 35S- jmt /ir- mje plants (*, p
    Figure Legend Snippet: In N. attenuata , OS Ms induced higher NaLRRK1 transcript levels in 35S- jmt /ir- mje plants than in WT plants. ( a ) diagram shows the JA metabolic flux in WT (left) and 35S- jmt /ir- mje plants (right). ( b ) in comparison to WT plants, 35S- jmt /ir- mje plants have reduced level of OS Ms -induced JA-Ile. ( c ) the transcript levels of NaLRRK1 are higher in 35S- jmt /ir- mje plants than in WT plants. In both panel b and c, filled and unfilled bars indicate 35S jmt /ir- mje and WT plants, respectively. Data are presented as means ± SEM. Rosette stage N. attenuata plants were wounded and 20 µL of OS Ms was immediately applied to the wounds. Leaf samples were collected at 1 hr after elicitations. Asterisk indicates significant differences between WT and 35S- jmt /ir- mje plants (*, p

    Techniques Used: Mass Spectrometry

    NaLRRK1 transcript abundance was successfully reduced in VIGS- NaLRRK1 plants in comparison to controls. a and b NaLRRK1 transcript levels in leaves that were undamaged ( a ) and or treated with wounding + FAC ( b ) For treated leaves, samples were collected 0.5 hr after elicitation. For each treatment, 8 biological replicates were analyzed. No morphological differences between VIGS-EV and VIGS- NaLRRK1 plants at the rosette-stage of growth were observed. The VIGS-NaPDS was used as a positive control for the VIGS process. Asterisks indicate significant differences between VIGS-EV and VIGS- NaLRRK1 plants (***, p
    Figure Legend Snippet: NaLRRK1 transcript abundance was successfully reduced in VIGS- NaLRRK1 plants in comparison to controls. a and b NaLRRK1 transcript levels in leaves that were undamaged ( a ) and or treated with wounding + FAC ( b ) For treated leaves, samples were collected 0.5 hr after elicitation. For each treatment, 8 biological replicates were analyzed. No morphological differences between VIGS-EV and VIGS- NaLRRK1 plants at the rosette-stage of growth were observed. The VIGS-NaPDS was used as a positive control for the VIGS process. Asterisks indicate significant differences between VIGS-EV and VIGS- NaLRRK1 plants (***, p

    Techniques Used: Positive Control

    VIGS- NaLRRK1 plants accumulated higher levels of FAC induced soluble sugars and invertase activity in comparison to control. ( a–d ) concentrations of different soluble sugars in VIGS-EV and VIGS- NaLRRK1 plants. ( e ) the activity of soluble invertase in VIGS-EV and VIGS- NaLRRK1 plants. Leaves were collected at 24 hr after wounding and FAC elicitations. Asterisks indicate significant differences between VIGS-EV and VIGS- NaLRRK1 plants (*, p
    Figure Legend Snippet: VIGS- NaLRRK1 plants accumulated higher levels of FAC induced soluble sugars and invertase activity in comparison to control. ( a–d ) concentrations of different soluble sugars in VIGS-EV and VIGS- NaLRRK1 plants. ( e ) the activity of soluble invertase in VIGS-EV and VIGS- NaLRRK1 plants. Leaves were collected at 24 hr after wounding and FAC elicitations. Asterisks indicate significant differences between VIGS-EV and VIGS- NaLRRK1 plants (*, p

    Techniques Used: Activity Assay

    25) Product Images from "The Functional Roles of the MDM2 Splice Variants P2-MDM2-10 and MDM2-∆5 in Breast Cancer Cells"

    Article Title: The Functional Roles of the MDM2 Splice Variants P2-MDM2-10 and MDM2-∆5 in Breast Cancer Cells

    Journal: Translational Oncology

    doi: 10.1016/j.tranon.2017.07.006

    Induction of apoptosis and senescence in MCF-7 cells. (A) Bars illustrate the percentage of apoptotic cells after transfection with pCMV (TOPO-Control), MDM2-FL, and the splice variants, untreated (purple bars) or treated with 1 μM doxorubicin (green bars) and analyzed by Annexin V assay 24 hours post transfection. The bars represent the cells that are both early apoptotic and apoptotic. The experiment was repeated in triplicate with three independent transfections. (B) Graphs illustrate the percentage of senescent cells after transfection with pCMV (TOPO-Control), P2-MDM2-10, and MDM2-Δ5. Untreated (purple bars) or treated with 0.25 μM doxorubicin (green bars) analyzed by β-galactosidase assay 8 days posttransfection. The experiment was repeated in triplicate with three independent transfections.
    Figure Legend Snippet: Induction of apoptosis and senescence in MCF-7 cells. (A) Bars illustrate the percentage of apoptotic cells after transfection with pCMV (TOPO-Control), MDM2-FL, and the splice variants, untreated (purple bars) or treated with 1 μM doxorubicin (green bars) and analyzed by Annexin V assay 24 hours post transfection. The bars represent the cells that are both early apoptotic and apoptotic. The experiment was repeated in triplicate with three independent transfections. (B) Graphs illustrate the percentage of senescent cells after transfection with pCMV (TOPO-Control), P2-MDM2-10, and MDM2-Δ5. Untreated (purple bars) or treated with 0.25 μM doxorubicin (green bars) analyzed by β-galactosidase assay 8 days posttransfection. The experiment was repeated in triplicate with three independent transfections.

    Techniques Used: Transfection, Annexin V Assay

    26) Product Images from "Development of a Markerless Knockout Method for Actinobacillus succinogenes"

    Article Title: Development of a Markerless Knockout Method for Actinobacillus succinogenes

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.00492-14

    Physical maps of pCR2.1-Δ pflB :: icd and its truncated constructs pCR2.1-Δ pflB :: icd -600, pCR2.1-Δ pflB :: icd -400, and pCR2.1-Δ pflB :: icd -200. A, AscI; black triangles, primers; vertical arrows, USS sequences.
    Figure Legend Snippet: Physical maps of pCR2.1-Δ pflB :: icd and its truncated constructs pCR2.1-Δ pflB :: icd -600, pCR2.1-Δ pflB :: icd -400, and pCR2.1-Δ pflB :: icd -200. A, AscI; black triangles, primers; vertical arrows, USS sequences.

    Techniques Used: Construct

    27) Product Images from "Identification and characterization of the first pectin methylesterase gene discovered in the root lesion nematode Pratylenchus penetrans"

    Article Title: Identification and characterization of the first pectin methylesterase gene discovered in the root lesion nematode Pratylenchus penetrans

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0212540

    Molecular characterization of the pectin methylesterase ( Pp-pme ) of Pratylenchus penetrans . (A) Amplicons of both genomic (2,501 bp) and cDNA coding (987 bp) sequences of Pp-pme , respectively. (B) Schematic representation of the Pp-pme gene structure. Relative positions and respective sizes of the exons are indicated by dark boxes and introns by lines.
    Figure Legend Snippet: Molecular characterization of the pectin methylesterase ( Pp-pme ) of Pratylenchus penetrans . (A) Amplicons of both genomic (2,501 bp) and cDNA coding (987 bp) sequences of Pp-pme , respectively. (B) Schematic representation of the Pp-pme gene structure. Relative positions and respective sizes of the exons are indicated by dark boxes and introns by lines.

    Techniques Used:

    28) 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

    29) Product Images from "Expression of endoglucanases in Pichia pastoris under control of the GAP promoter"

    Article Title: Expression of endoglucanases in Pichia pastoris under control of the GAP promoter

    Journal: Microbial Cell Factories

    doi: 10.1186/1475-2859-13-57

    Endoglucanase production in 3-liter bioreactors with fed-batch cultivation. One clone of each PichiaPink™ recombinant strain was grown in two parallel 3-l fermenters. The SDS-PAGE figures show the accumulation of A) Af Cel12A and B) Ta Cel5A. Sample lanes are marked with the age of the culture in hours for each fermenter. Reactor B of AOX1/AfCel12A is missing from the gel as cells stopped growing in the batch phase. The bands appearing at approx. 24 kDa (panel A) represent Af Cel12A; the bands appearing at approx. 37 kDa represent Ta Cel5A (panel B).
    Figure Legend Snippet: Endoglucanase production in 3-liter bioreactors with fed-batch cultivation. One clone of each PichiaPink™ recombinant strain was grown in two parallel 3-l fermenters. The SDS-PAGE figures show the accumulation of A) Af Cel12A and B) Ta Cel5A. Sample lanes are marked with the age of the culture in hours for each fermenter. Reactor B of AOX1/AfCel12A is missing from the gel as cells stopped growing in the batch phase. The bands appearing at approx. 24 kDa (panel A) represent Af Cel12A; the bands appearing at approx. 37 kDa represent Ta Cel5A (panel B).

    Techniques Used: Recombinant, SDS Page

    Endoglucanase activity in culture supernatants obtained during tube-scale screening of transformants. Culture supernatants were collected from the AOX1 strains 48 hours after induction, and from the GAP strains after 48 hours of incubation (see Methods for more details). A) Endoglucanase activity was measured on filter paper by incubating 10 μl culture supernatant with 40 μl of a suspension of phosphoric acid swollen filter paper (1% w/v) and 10 μl 50 mM Na-citrate buffer (pH 5.0) for 1 hour at 50°C. B) Endoglucanase activity was measured on carboxymethylcellulose by incubating 50 μl appropriately diluted culture supernatant with 450 μl 1% (w/v) carboxymethylcellulose in 50 mM Na-citrate buffer (pH 5.0) for 10 min at 50°C. Enzyme activities were assessed by measuring formation of reducing sugars. Filter paper activity is expressed as percentage of substrate conversion; CMCase activity is expressed as nkat/ml. The activity values were background corrected by measuring reducing sugars present in the culture supernatants. Since culture supernatants of PichiaPink TM transformed with the empty vector, lacking endoglucanases, showed no activity, all activity can be attributed to the recombinant endoglucanases. The data are means from triplicate experiments; error bars reflect standard deviations. The clone number is indicated in the bars.
    Figure Legend Snippet: Endoglucanase activity in culture supernatants obtained during tube-scale screening of transformants. Culture supernatants were collected from the AOX1 strains 48 hours after induction, and from the GAP strains after 48 hours of incubation (see Methods for more details). A) Endoglucanase activity was measured on filter paper by incubating 10 μl culture supernatant with 40 μl of a suspension of phosphoric acid swollen filter paper (1% w/v) and 10 μl 50 mM Na-citrate buffer (pH 5.0) for 1 hour at 50°C. B) Endoglucanase activity was measured on carboxymethylcellulose by incubating 50 μl appropriately diluted culture supernatant with 450 μl 1% (w/v) carboxymethylcellulose in 50 mM Na-citrate buffer (pH 5.0) for 10 min at 50°C. Enzyme activities were assessed by measuring formation of reducing sugars. Filter paper activity is expressed as percentage of substrate conversion; CMCase activity is expressed as nkat/ml. The activity values were background corrected by measuring reducing sugars present in the culture supernatants. Since culture supernatants of PichiaPink TM transformed with the empty vector, lacking endoglucanases, showed no activity, all activity can be attributed to the recombinant endoglucanases. The data are means from triplicate experiments; error bars reflect standard deviations. The clone number is indicated in the bars.

    Techniques Used: Activity Assay, Incubation, Transformation Assay, Plasmid Preparation, Recombinant

    30) 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

    31) Product Images from "In vivo dissection of the chromosome condensation machinery"

    Article Title: In vivo dissection of the chromosome condensation machinery

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200109056

    Topo II and phospho-H3 are not essential for rDNA condensation. CH325 ( top2-4 ), JHY90 ( WT ), JHY91 ( H3-S10A ), and JHY93 ( H3-S10,28A ) cultures were synchronized in G1, and released into a Nz block either at 37°C (top2-4) or 23°C. After rearrest in M phase, cells were fixed and processed for rDNA FISH. rDNA loops were scored as condensed. Greater than 100 nuclei/sample were scored. Data for the condensins is from Fig. 2 and is shown for comparison.
    Figure Legend Snippet: Topo II and phospho-H3 are not essential for rDNA condensation. CH325 ( top2-4 ), JHY90 ( WT ), JHY91 ( H3-S10A ), and JHY93 ( H3-S10,28A ) cultures were synchronized in G1, and released into a Nz block either at 37°C (top2-4) or 23°C. After rearrest in M phase, cells were fixed and processed for rDNA FISH. rDNA loops were scored as condensed. Greater than 100 nuclei/sample were scored. Data for the condensins is from Fig. 2 and is shown for comparison.

    Techniques Used: Blocking Assay, Fluorescence In Situ Hybridization

    32) Product Images from "Single-cell analysis of the fate of c-kit-positive bone marrow cells"

    Article Title: Single-cell analysis of the fate of c-kit-positive bone marrow cells

    Journal: NPJ Regenerative Medicine

    doi: 10.1038/s41536-017-0032-1

    c-kit-BMCs acquire distinct cardiac cell phenotypes in vivo. a Representative scatter plots illustrating the expression of c-kit, Thy1.2 and CD31 in cardiac cell populations isolated from c-kit-BMC-treated infarcted hearts. The percentage of positive cells is indicated. CTRL: isotype control; SSC: side scatter. b Transcripts for α-myosin heavy chain ( Myh6 ), c-kit, CD31, collagen type III α-1 ( Col3a1 ) and β-2 microglobulin ( B2M ) in isolated cardiomyocytes (Myo), c-kit-BMCs (c-kit), endothelial cells (ECs) and fibroblasts (Fbl). Myocardium (first lane, MC) was used as control. bp: base pairs. c Isolated cardiomyocytes expressing α-sarcomeric actin (α-SA, red), ECs expressing von Willebrand factor (vWF, yellow) and fibroblasts expressing procollagen (Pro-Col, green) are shown. Quantitative data are presented as mean ± SD. Scale bars: Left and central panels = 50 µm; Right panel = 20 µm. d PCR products run on agarose gel correspond to the sites of integration of the viral genome in the DNA of c-kit-BMCs and myocytes. These images correspond to representative examples of experiments conducted in 8 mice. The upper band shows the pCR4-TOPO TA vector. Molecular mass: 100 bp incremental ladders
    Figure Legend Snippet: c-kit-BMCs acquire distinct cardiac cell phenotypes in vivo. a Representative scatter plots illustrating the expression of c-kit, Thy1.2 and CD31 in cardiac cell populations isolated from c-kit-BMC-treated infarcted hearts. The percentage of positive cells is indicated. CTRL: isotype control; SSC: side scatter. b Transcripts for α-myosin heavy chain ( Myh6 ), c-kit, CD31, collagen type III α-1 ( Col3a1 ) and β-2 microglobulin ( B2M ) in isolated cardiomyocytes (Myo), c-kit-BMCs (c-kit), endothelial cells (ECs) and fibroblasts (Fbl). Myocardium (first lane, MC) was used as control. bp: base pairs. c Isolated cardiomyocytes expressing α-sarcomeric actin (α-SA, red), ECs expressing von Willebrand factor (vWF, yellow) and fibroblasts expressing procollagen (Pro-Col, green) are shown. Quantitative data are presented as mean ± SD. Scale bars: Left and central panels = 50 µm; Right panel = 20 µm. d PCR products run on agarose gel correspond to the sites of integration of the viral genome in the DNA of c-kit-BMCs and myocytes. These images correspond to representative examples of experiments conducted in 8 mice. The upper band shows the pCR4-TOPO TA vector. Molecular mass: 100 bp incremental ladders

    Techniques Used: In Vivo, Expressing, Isolation, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Mouse Assay, Plasmid Preparation

    33) Product Images from "CRISPR/Cas9‐mediated knockout of six glycosyltransferase genes in Nicotiana benthamiana for the production of recombinant proteins lacking β‐1,2‐xylose and core α‐1,3‐fucose"

    Article Title: CRISPR/Cas9‐mediated knockout of six glycosyltransferase genes in Nicotiana benthamiana for the production of recombinant proteins lacking β‐1,2‐xylose and core α‐1,3‐fucose

    Journal: Plant Biotechnology Journal

    doi: 10.1111/pbi.12981

    Sequencing and Western blot analysis for the T 2 lines X‐KO #34‐4‐4 and F‐KO #6‐10‐6 and the F 2 line FX‐KO #20‐4. (a) Mutations in XylT 1 and 2 of X‐KO #34‐4‐4 as identified by Sanger sequencing of PCR amplicons ( XylT 1) and TOPO‐cloned PCR products ( XylT 2). The corresponding wild‐type sequence is shown above, the gRNA target sequences are indicated by coloured boxes, and the PAM sequences are shown in bold. (b) Mutations in FucT 1, 2, 3 and 4 of F‐KO #6‐10‐6. The PAM‐distal mismatch between the gRNA and Fuc T 3 and 4 is highlighted in yellow. (c) Mutations in FucT 1‐4 and XylT 1 and 2 of FX‐KO #20‐4. Five of six genes carry homozygous mutations, with the exception of FucT 3 which has biallelic mutations. (d) Western blot of FX‐KO #20‐4 in comparison to N. benthamiana wild type, F‐KO #6‐10‐6 and X‐KO #34‐4‐4. ~10 µg of total soluble protein were loaded for each sample. Anti‐fucose blot: 1st antibody rabbit‐anti‐α1,3‐fucose (1 : 10 000), 2nd antibody goat‐anti‐rabbit H+L AP‐labelled (1 : 10 000, pre‐absorbed). Anti‐xylose blot: 1st antibody rabbit‐anti‐β1,2‐xylose (1 : 5000), 2nd antibody goat‐anti‐rabbit H+L AP‐labelled (1 : 10 000, pre‐absorbed).
    Figure Legend Snippet: Sequencing and Western blot analysis for the T 2 lines X‐KO #34‐4‐4 and F‐KO #6‐10‐6 and the F 2 line FX‐KO #20‐4. (a) Mutations in XylT 1 and 2 of X‐KO #34‐4‐4 as identified by Sanger sequencing of PCR amplicons ( XylT 1) and TOPO‐cloned PCR products ( XylT 2). The corresponding wild‐type sequence is shown above, the gRNA target sequences are indicated by coloured boxes, and the PAM sequences are shown in bold. (b) Mutations in FucT 1, 2, 3 and 4 of F‐KO #6‐10‐6. The PAM‐distal mismatch between the gRNA and Fuc T 3 and 4 is highlighted in yellow. (c) Mutations in FucT 1‐4 and XylT 1 and 2 of FX‐KO #20‐4. Five of six genes carry homozygous mutations, with the exception of FucT 3 which has biallelic mutations. (d) Western blot of FX‐KO #20‐4 in comparison to N. benthamiana wild type, F‐KO #6‐10‐6 and X‐KO #34‐4‐4. ~10 µg of total soluble protein were loaded for each sample. Anti‐fucose blot: 1st antibody rabbit‐anti‐α1,3‐fucose (1 : 10 000), 2nd antibody goat‐anti‐rabbit H+L AP‐labelled (1 : 10 000, pre‐absorbed). Anti‐xylose blot: 1st antibody rabbit‐anti‐β1,2‐xylose (1 : 5000), 2nd antibody goat‐anti‐rabbit H+L AP‐labelled (1 : 10 000, pre‐absorbed).

    Techniques Used: Sequencing, Western Blot, Polymerase Chain Reaction, Clone Assay

    34) Product Images from "Generation of cattle knockout for galactose‐α1,3‐galactose and N‐glycolylneuraminic acid antigens, et al. Generation of cattle knockout for galactose‐α1,3‐galactose and N‐glycolylneuraminic acid antigens"

    Article Title: Generation of cattle knockout for galactose‐α1,3‐galactose and N‐glycolylneuraminic acid antigens, et al. Generation of cattle knockout for galactose‐α1,3‐galactose and N‐glycolylneuraminic acid antigens

    Journal: Xenotransplantation

    doi: 10.1111/xen.12524

    Editing of GGTA1 and CMAH genes in male and female fibroblasts. A, Target sequences for selected sgRNAs and ss CMAH ‐STOP oligo sequence. For each bovine gene ( GGTA1 and CMAH ), target sequences are indicated on the respective exons recognized by the selected sgRNAs. PAM sequences are highlighted in blue. In the ss CMAH ‐STOP oligo sequence, the TAA (STOP) codon is highlighted in bold character; the Afl II restriction site is underlined. B, PCR analyses of female colonies. The results of the PCR analyses performed for the genomic characterization of the female colonies (A1, A2, A3, A4, A5 and A6) selected after Dynabeads sorting are reported as an example. Each colony was analysed for the GGTA1 gene (739 bp) and for the CMAH gene (225 bp). Resulting electrophoretic patterns determined directly that some colonies were characterized by visible Indels , creating bands different from the WT controls. This situation is clear for colonies A1 (double band), A2 (deletion) and A6 (deletion) in PCR analyses for the GGTA1 gene (°) and for colonies A1 (double band) and A5 (deletion) in PCR analyses for the CMAH gene (#). Resulting CMAH ‐PCR products were also digested with the Afl II restriction enzyme, detecting the alleles interested by the targeting event. Due to the introduction of a STOP codon (TAA) in the START position (ATG) of the CMAH gene, only the HDR‐ CMAH alleles will be cut by the restriction enzyme producing two lower bands (152 + 73 bp). A simple agarose electrophoresis enabled us to identify possible additional edited colonies detecting the STOP codon insertion (**) for colonies A2 and A6 and the single insertion (*) for colonies A3 and A4. In these last ones, the not targeted allele resulted uncut (225 bp) as the WT sample. For this reason, the final determination of the exact Indels , occurred in all the edited colonies, was determined by Sanger sequencing of the resulting TOPO TA E coli clones. 100 = 100 bp ladder (Thermo Fisher Scientific); A1, A2, A3, A4, A5 and A6 = transfected females colonies; WT = wild‐type female line; H 2 0 = Nucleases‐free water. C, Sequences alignments of colonies used for the SCNT. Sanger sequencing outlining the mutations affecting the GGTA1 and the CMAH genes of colonies selected for the SCNT step. For the GGTA1 gene, the exon 9 was used as reference for the male colonies and a PCR product including the exon 4 was used for the female ones. In both cases, deletions of different lengths were obtained (Table S1 ). For the CMAH gene, all edited alleles of the edited colonies were aligned using as reference a PCR product including the exon 2 sequence. In this case, in both lines, we were able to determine the TAA substitution, as result of the targeting event mediated by the site‐specific cut, produced by the CRISPR/Cas9 system driven by the sgRNA bt CMAH cr1
    Figure Legend Snippet: Editing of GGTA1 and CMAH genes in male and female fibroblasts. A, Target sequences for selected sgRNAs and ss CMAH ‐STOP oligo sequence. For each bovine gene ( GGTA1 and CMAH ), target sequences are indicated on the respective exons recognized by the selected sgRNAs. PAM sequences are highlighted in blue. In the ss CMAH ‐STOP oligo sequence, the TAA (STOP) codon is highlighted in bold character; the Afl II restriction site is underlined. B, PCR analyses of female colonies. The results of the PCR analyses performed for the genomic characterization of the female colonies (A1, A2, A3, A4, A5 and A6) selected after Dynabeads sorting are reported as an example. Each colony was analysed for the GGTA1 gene (739 bp) and for the CMAH gene (225 bp). Resulting electrophoretic patterns determined directly that some colonies were characterized by visible Indels , creating bands different from the WT controls. This situation is clear for colonies A1 (double band), A2 (deletion) and A6 (deletion) in PCR analyses for the GGTA1 gene (°) and for colonies A1 (double band) and A5 (deletion) in PCR analyses for the CMAH gene (#). Resulting CMAH ‐PCR products were also digested with the Afl II restriction enzyme, detecting the alleles interested by the targeting event. Due to the introduction of a STOP codon (TAA) in the START position (ATG) of the CMAH gene, only the HDR‐ CMAH alleles will be cut by the restriction enzyme producing two lower bands (152 + 73 bp). A simple agarose electrophoresis enabled us to identify possible additional edited colonies detecting the STOP codon insertion (**) for colonies A2 and A6 and the single insertion (*) for colonies A3 and A4. In these last ones, the not targeted allele resulted uncut (225 bp) as the WT sample. For this reason, the final determination of the exact Indels , occurred in all the edited colonies, was determined by Sanger sequencing of the resulting TOPO TA E coli clones. 100 = 100 bp ladder (Thermo Fisher Scientific); A1, A2, A3, A4, A5 and A6 = transfected females colonies; WT = wild‐type female line; H 2 0 = Nucleases‐free water. C, Sequences alignments of colonies used for the SCNT. Sanger sequencing outlining the mutations affecting the GGTA1 and the CMAH genes of colonies selected for the SCNT step. For the GGTA1 gene, the exon 9 was used as reference for the male colonies and a PCR product including the exon 4 was used for the female ones. In both cases, deletions of different lengths were obtained (Table S1 ). For the CMAH gene, all edited alleles of the edited colonies were aligned using as reference a PCR product including the exon 2 sequence. In this case, in both lines, we were able to determine the TAA substitution, as result of the targeting event mediated by the site‐specific cut, produced by the CRISPR/Cas9 system driven by the sgRNA bt CMAH cr1

    Techniques Used: Sequencing, Polymerase Chain Reaction, Electrophoresis, Clone Assay, Transfection, Produced, CRISPR

    35) Product Images from "Inactivation of class II transactivator by DNA methylation and histone deacetylation associated with absence of HLA-DR induction by interferon-γ in haematopoietic tumour cells"

    Article Title: Inactivation of class II transactivator by DNA methylation and histone deacetylation associated with absence of HLA-DR induction by interferon-γ in haematopoietic tumour cells

    Journal: British Journal of Cancer

    doi: 10.1038/sj.bjc.6601602

    Bisulphite sequencing of CIITA. Amplified PCR products were cloned into pCR4 vector using a TOPO-TA cloning Kit (Invitrogen) and plasmid DNA was purified. Sequencing reaction was performed using a Big-Dye terminator Kit (Applied Biosystems) and electrophoresed using an ABI3100 system (Applied Biosystems). CpG sites are shown above. Methylated alleles are shown as solid circles; unmethylated alleles as shown as open circles.
    Figure Legend Snippet: Bisulphite sequencing of CIITA. Amplified PCR products were cloned into pCR4 vector using a TOPO-TA cloning Kit (Invitrogen) and plasmid DNA was purified. Sequencing reaction was performed using a Big-Dye terminator Kit (Applied Biosystems) and electrophoresed using an ABI3100 system (Applied Biosystems). CpG sites are shown above. Methylated alleles are shown as solid circles; unmethylated alleles as shown as open circles.

    Techniques Used: Bisulfite Sequencing, Amplification, Polymerase Chain Reaction, Clone Assay, Plasmid Preparation, TA Cloning, Purification, Sequencing, Methylation

    36) 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

    37) Product Images from "Cloning and Characterization of the Gene Encoding the Glutamate Dehydrogenase of Streptococcus suis Serotype 2"

    Article Title: Cloning and Characterization of the Gene Encoding the Glutamate Dehydrogenase of Streptococcus suis Serotype 2

    Journal: Clinical and Diagnostic Laboratory Immunology

    doi: 10.1128/CDLI.8.2.251-257.2001

    Overexpression and purification of the 45-kDa recombinant GDH protein. The protein was overexpressed and purified as described in Materials and Methods. An SDS-10% polyacrylamide gel stained with Coomassie brilliant blue R-250 is shown. Lanes: M, rainbow molecular size marker in kilodaltons; 1, whole-cell lysate of pOT411 transformant of E. coli TOP10 uninduced; 2, whole-cell lysate of E. coli transformed with pOT411 and induced with arabinose; 3 and 4, different amounts of the recombinant protein purified from pOT411 transformant of E. coli TOP10.
    Figure Legend Snippet: Overexpression and purification of the 45-kDa recombinant GDH protein. The protein was overexpressed and purified as described in Materials and Methods. An SDS-10% polyacrylamide gel stained with Coomassie brilliant blue R-250 is shown. Lanes: M, rainbow molecular size marker in kilodaltons; 1, whole-cell lysate of pOT411 transformant of E. coli TOP10 uninduced; 2, whole-cell lysate of E. coli transformed with pOT411 and induced with arabinose; 3 and 4, different amounts of the recombinant protein purified from pOT411 transformant of E. coli TOP10.

    Techniques Used: Over Expression, Purification, Recombinant, Staining, Marker, Transformation Assay

    38) Product Images from "REPLACR-mutagenesis, a one-step method for site-directed mutagenesis by recombineering"

    Article Title: REPLACR-mutagenesis, a one-step method for site-directed mutagenesis by recombineering

    Journal: Scientific Reports

    doi: 10.1038/srep19121

    Efficiency of REPLACR-mutagenesis. ( a ) The effect of homology at the ends of PCR products is plotted against the achieved efficiencies with REPLACR-mutagenesis. The efficiency of mutagenesis increases with increasing homology, where a 2 bp homology is insufficient to yield any correct products while a 17 bp homology gives the highest efficiency at 84%. ( b ) The same PCR products with 14 bp and 17 bp homology were used with two commercial kits (Gibson Assembly and GeneArt Seamless cloning) and the achieved efficiencies were compared with REPLACR-mutagenesis. REPLACR-mutagenesis is least efficient among the three methods when the PCR products have only 14 bp homology, however with the recommended 17 bp homology at the ends of PCR products, comparable efficiencies for all the methods can be observed. The data is presented as mean ± standard error of mean (SEM) of three independent repeats.
    Figure Legend Snippet: Efficiency of REPLACR-mutagenesis. ( a ) The effect of homology at the ends of PCR products is plotted against the achieved efficiencies with REPLACR-mutagenesis. The efficiency of mutagenesis increases with increasing homology, where a 2 bp homology is insufficient to yield any correct products while a 17 bp homology gives the highest efficiency at 84%. ( b ) The same PCR products with 14 bp and 17 bp homology were used with two commercial kits (Gibson Assembly and GeneArt Seamless cloning) and the achieved efficiencies were compared with REPLACR-mutagenesis. REPLACR-mutagenesis is least efficient among the three methods when the PCR products have only 14 bp homology, however with the recommended 17 bp homology at the ends of PCR products, comparable efficiencies for all the methods can be observed. The data is presented as mean ± standard error of mean (SEM) of three independent repeats.

    Techniques Used: Mutagenesis, Polymerase Chain Reaction, Clone Assay

    39) Product Images from "REPLACR-mutagenesis, a one-step method for site-directed mutagenesis by recombineering"

    Article Title: REPLACR-mutagenesis, a one-step method for site-directed mutagenesis by recombineering

    Journal: Scientific Reports

    doi: 10.1038/srep19121

    Efficiency of REPLACR-mutagenesis. ( a ) The effect of homology at the ends of PCR products is plotted against the achieved efficiencies with REPLACR-mutagenesis. The efficiency of mutagenesis increases with increasing homology, where a 2 bp homology is insufficient to yield any correct products while a 17 bp homology gives the highest efficiency at 84%. ( b ) The same PCR products with 14 bp and 17 bp homology were used with two commercial kits (Gibson Assembly and GeneArt Seamless cloning) and the achieved efficiencies were compared with REPLACR-mutagenesis. REPLACR-mutagenesis is least efficient among the three methods when the PCR products have only 14 bp homology, however with the recommended 17 bp homology at the ends of PCR products, comparable efficiencies for all the methods can be observed. The data is presented as mean ± standard error of mean (SEM) of three independent repeats.
    Figure Legend Snippet: Efficiency of REPLACR-mutagenesis. ( a ) The effect of homology at the ends of PCR products is plotted against the achieved efficiencies with REPLACR-mutagenesis. The efficiency of mutagenesis increases with increasing homology, where a 2 bp homology is insufficient to yield any correct products while a 17 bp homology gives the highest efficiency at 84%. ( b ) The same PCR products with 14 bp and 17 bp homology were used with two commercial kits (Gibson Assembly and GeneArt Seamless cloning) and the achieved efficiencies were compared with REPLACR-mutagenesis. REPLACR-mutagenesis is least efficient among the three methods when the PCR products have only 14 bp homology, however with the recommended 17 bp homology at the ends of PCR products, comparable efficiencies for all the methods can be observed. The data is presented as mean ± standard error of mean (SEM) of three independent repeats.

    Techniques Used: Mutagenesis, Polymerase Chain Reaction, Clone Assay

    40) Product Images from "Duplication and concerted evolution of MiSp-encoding genes underlie the material properties of minor ampullate silks of cobweb weaving spiders"

    Article Title: Duplication and concerted evolution of MiSp-encoding genes underlie the material properties of minor ampullate silks of cobweb weaving spiders

    Journal: BMC Evolutionary Biology

    doi: 10.1186/s12862-017-0927-x

    Neighbor joining tree for N-terminal MiSp encoding sequences demonstrates variation within and among species. Variants are shown for L. hesperus ( Lh ) and TOPO clones from L. tredecimguttatus ( Lt ), L. geometricus ( Lg ), and S. grossa ( Sg ). Units are number of substitutions. Latrodectus clones are arbitrarily numbered, except that Lt M1-7 and Lg M1-5 resulted from amplification with primers designed from L. hesperus , while higher clone numbers resulted from amplification with species-specific primers. S. grossa TOPO clones resulted from two separate PCR reactions, which are indicated here as “A#” and “B#”. Completely sequenced clones are indicated by bolded names. See Table 1 and Additional file 1 : Table S4 for accession numbers. Boxes indicate distinct clusters identified in neighbor joining trees based on N-terminal sequences including adjacent repetitive sequences and in C-terminal and adjacent repetitive region encoding sequences (Additional file 1 : Figures S2 and S3)
    Figure Legend Snippet: Neighbor joining tree for N-terminal MiSp encoding sequences demonstrates variation within and among species. Variants are shown for L. hesperus ( Lh ) and TOPO clones from L. tredecimguttatus ( Lt ), L. geometricus ( Lg ), and S. grossa ( Sg ). Units are number of substitutions. Latrodectus clones are arbitrarily numbered, except that Lt M1-7 and Lg M1-5 resulted from amplification with primers designed from L. hesperus , while higher clone numbers resulted from amplification with species-specific primers. S. grossa TOPO clones resulted from two separate PCR reactions, which are indicated here as “A#” and “B#”. Completely sequenced clones are indicated by bolded names. See Table 1 and Additional file 1 : Table S4 for accession numbers. Boxes indicate distinct clusters identified in neighbor joining trees based on N-terminal sequences including adjacent repetitive sequences and in C-terminal and adjacent repetitive region encoding sequences (Additional file 1 : Figures S2 and S3)

    Techniques Used: Clone Assay, Amplification, Polymerase Chain Reaction

    Arrangement of amino acid motifs in MiSp sequences of cobweb and orb-web weaving spiders. Latrodectus and S. grossa accession numbers are in Table 1 . Lh , L. hesperus ; Lt , L. tredecimguttatus ; Lg , L. geometricus ; Sg , S. grossa ; Pt , P. tepidariorum based on combining the incompletely assembled MiSp-encoding region from Scaffold 853 of the i5K genome with an ~2.3 kb TOPO-cloned PCR product, KX584004; Av , A. ventricosus, JX513956.1 [ 30 ]. Lengths of missing amino acid sequences in Pt MiSp_v1 are based on the length of gaps in Scaffold 853 of the i5K genome, assuming no introns are present within those gaps. The last gap was predicted to be 1782 bp long (594 aa if no intron) in Scaffold 853, but our TOPO clone added 2236 bp, including a 1175 bp intron, which was longer than the predicted gap. The repetitive encoding region at the 3’ end of our TOPO clone did not overlap with the encoding region present in the i5K genome prior to the last gap
    Figure Legend Snippet: Arrangement of amino acid motifs in MiSp sequences of cobweb and orb-web weaving spiders. Latrodectus and S. grossa accession numbers are in Table 1 . Lh , L. hesperus ; Lt , L. tredecimguttatus ; Lg , L. geometricus ; Sg , S. grossa ; Pt , P. tepidariorum based on combining the incompletely assembled MiSp-encoding region from Scaffold 853 of the i5K genome with an ~2.3 kb TOPO-cloned PCR product, KX584004; Av , A. ventricosus, JX513956.1 [ 30 ]. Lengths of missing amino acid sequences in Pt MiSp_v1 are based on the length of gaps in Scaffold 853 of the i5K genome, assuming no introns are present within those gaps. The last gap was predicted to be 1782 bp long (594 aa if no intron) in Scaffold 853, but our TOPO clone added 2236 bp, including a 1175 bp intron, which was longer than the predicted gap. The repetitive encoding region at the 3’ end of our TOPO clone did not overlap with the encoding region present in the i5K genome prior to the last gap

    Techniques Used: Clone Assay, Polymerase Chain Reaction

    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′.

    Article Title: Effect of Bottlenecking on Evolution of the Nonstructural Protein 3 Gene of Hepatitis C Virus during Sexually Transmitted Acute Resolving Infection
    Article Snippet: .. Nested PCR products from samples A-10/1998 and B-9/1998 were cloned into the pcR-4Blunt TOPO plasmid by using the Zero Blunt TOPO PCR cloning kit in TOP10 Escherichia coli cells (Invitrogen, Carlsbad, CA), taking advantage of the fact that the Pfu DNA polymerase is devoid of terminal deoxynucleotidyl transferase activity and generates blunt-ended PCR products exclusively. .. Overlapping primers in both directions (up and down) (Fig. ) were used to obtain the complete NS3 sequence of 1,893 nucleotides from either PCR products or cloned fragments.

    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′.

    Article Title: Effect of Bottlenecking on Evolution of the Nonstructural Protein 3 Gene of Hepatitis C Virus during Sexually Transmitted Acute Resolving Infection
    Article Snippet: .. Nested PCR products from samples A-10/1998 and B-9/1998 were cloned into the pcR-4Blunt TOPO plasmid by using the Zero Blunt TOPO PCR cloning kit in TOP10 Escherichia coli cells (Invitrogen, Carlsbad, CA), taking advantage of the fact that the Pfu DNA polymerase is devoid of terminal deoxynucleotidyl transferase activity and generates blunt-ended PCR products exclusively. .. Overlapping primers in both directions (up and down) (Fig. ) were used to obtain the complete NS3 sequence of 1,893 nucleotides from either PCR products or cloned fragments.

    Activity Assay:

    Article Title: Effect of Bottlenecking on Evolution of the Nonstructural Protein 3 Gene of Hepatitis C Virus during Sexually Transmitted Acute Resolving Infection
    Article Snippet: .. Nested PCR products from samples A-10/1998 and B-9/1998 were cloned into the pcR-4Blunt TOPO plasmid by using the Zero Blunt TOPO PCR cloning kit in TOP10 Escherichia coli cells (Invitrogen, Carlsbad, CA), taking advantage of the fact that the Pfu DNA polymerase is devoid of terminal deoxynucleotidyl transferase activity and generates blunt-ended PCR products exclusively. .. Overlapping primers in both directions (up and down) (Fig. ) were used to obtain the complete NS3 sequence of 1,893 nucleotides from either PCR products or cloned fragments.

    Nested PCR:

    Article Title: Effect of Bottlenecking on Evolution of the Nonstructural Protein 3 Gene of Hepatitis C Virus during Sexually Transmitted Acute Resolving Infection
    Article Snippet: .. Nested PCR products from samples A-10/1998 and B-9/1998 were cloned into the pcR-4Blunt TOPO plasmid by using the Zero Blunt TOPO PCR cloning kit in TOP10 Escherichia coli cells (Invitrogen, Carlsbad, CA), taking advantage of the fact that the Pfu DNA polymerase is devoid of terminal deoxynucleotidyl transferase activity and generates blunt-ended PCR products exclusively. .. Overlapping primers in both directions (up and down) (Fig. ) were used to obtain the complete NS3 sequence of 1,893 nucleotides from either PCR products or cloned fragments.

    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′.

    Article Title: Effect of Bottlenecking on Evolution of the Nonstructural Protein 3 Gene of Hepatitis C Virus during Sexually Transmitted Acute Resolving Infection
    Article Snippet: .. Nested PCR products from samples A-10/1998 and B-9/1998 were cloned into the pcR-4Blunt TOPO plasmid by using the Zero Blunt TOPO PCR cloning kit in TOP10 Escherichia coli cells (Invitrogen, Carlsbad, CA), taking advantage of the fact that the Pfu DNA polymerase is devoid of terminal deoxynucleotidyl transferase activity and generates blunt-ended PCR products exclusively. .. Overlapping primers in both directions (up and down) (Fig. ) were used to obtain the complete NS3 sequence of 1,893 nucleotides from either PCR products or cloned fragments.

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    Thermo Fisher top10 competent e coli
    Top10 Competent E Coli, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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