cosmid psnogaori  (New England Biolabs)


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    Name:
    Exonuclease III E coli
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    Exonuclease III E coli 25 000 units
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    m0206l
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    Exonucleases
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    New England Biolabs cosmid psnogaori
    Exonuclease III E coli
    Exonuclease III E coli 25 000 units
    https://www.bioz.com/result/cosmid psnogaori/product/New England Biolabs
    Average 90 stars, based on 5084 article reviews
    Price from $9.99 to $1999.99
    cosmid psnogaori - by Bioz Stars, 2020-09
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    Images

    1) Product Images from "Divergent non-heme iron enzymes in the nogalamycin biosynthetic pathway"

    Article Title: Divergent non-heme iron enzymes in the nogalamycin biosynthetic pathway

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

    doi: 10.1073/pnas.1525034113

    The nogalamycin gene cluster and the biosynthetic steps for anthracyclines investigated in this study. The end product of the pathway nogalamycin ( 1 ) is produced by WT Streptomyces nogalater whereas pathway engineering led to the isolation of compounds 2 – 7 from the heterologous host S. albus , and compounds 8 and 9 were generated using enzymatic synthesis. The core of the gene cluster (white genes) has been cloned into cosmid pSnogaori for production of 2 whereas genes near the boundaries of the cluster are marked in black. Genes encoding SnoN and SnoK are shown in purple and gray, respectively.
    Figure Legend Snippet: The nogalamycin gene cluster and the biosynthetic steps for anthracyclines investigated in this study. The end product of the pathway nogalamycin ( 1 ) is produced by WT Streptomyces nogalater whereas pathway engineering led to the isolation of compounds 2 – 7 from the heterologous host S. albus , and compounds 8 and 9 were generated using enzymatic synthesis. The core of the gene cluster (white genes) has been cloned into cosmid pSnogaori for production of 2 whereas genes near the boundaries of the cluster are marked in black. Genes encoding SnoN and SnoK are shown in purple and gray, respectively.

    Techniques Used: Produced, Isolation, Generated, Clone Assay

    2) Product Images from "Interrogating and Predicting Tolerated Sequence Diversity in Protein Folds: Application to E. elaterium Trypsin Inhibitor-II Cystine-Knot Miniprotein"

    Article Title: Interrogating and Predicting Tolerated Sequence Diversity in Protein Folds: Application to E. elaterium Trypsin Inhibitor-II Cystine-Knot Miniprotein

    Journal: PLoS Computational Biology

    doi: 10.1371/journal.pcbi.1000499

    Trypsin-binding levels of EETI loop 3 predicted and randomly-generated clones. Motif-filtered clones (dark grey), least-similar clones (light grey), randomly generated clones (white), negative control (potato carboxypeptidase inhibitor knottin, black), and EETIwt (striped) were individually displayed on the surface of yeast and analyzed by flow cytometry. Predicted clones are preceded with a ‘p’ while randomly-generated clones are preceded with an ‘r.’ Protein expression levels were quantified by immunofluorescence staining of the cMyc epitope tag. Retention of the knottin fold was determined by binding of fluorescently-labeled trypsin (25 nM). Trypsin binding levels were adjusted to account for differences in protein expression levels and then normalized to the trypsin-binding level of EETIwt. Trypsin-binding experiments were performed in triplicate and error bars denote standard deviations. Predicted clones showing statistically significant differences in trypsin binding levels compared to EETIwt are marked with an asterisk (*) or a double asterisk (**) to indicate lower and higher binding levels, respectively.
    Figure Legend Snippet: Trypsin-binding levels of EETI loop 3 predicted and randomly-generated clones. Motif-filtered clones (dark grey), least-similar clones (light grey), randomly generated clones (white), negative control (potato carboxypeptidase inhibitor knottin, black), and EETIwt (striped) were individually displayed on the surface of yeast and analyzed by flow cytometry. Predicted clones are preceded with a ‘p’ while randomly-generated clones are preceded with an ‘r.’ Protein expression levels were quantified by immunofluorescence staining of the cMyc epitope tag. Retention of the knottin fold was determined by binding of fluorescently-labeled trypsin (25 nM). Trypsin binding levels were adjusted to account for differences in protein expression levels and then normalized to the trypsin-binding level of EETIwt. Trypsin-binding experiments were performed in triplicate and error bars denote standard deviations. Predicted clones showing statistically significant differences in trypsin binding levels compared to EETIwt are marked with an asterisk (*) or a double asterisk (**) to indicate lower and higher binding levels, respectively.

    Techniques Used: Binding Assay, Generated, Clone Assay, Negative Control, Flow Cytometry, Cytometry, Expressing, Immunofluorescence, Staining, Labeling

    Cladograms of predicted EETI loop 3 clones tested for their abilities to bind trypsin. Select predicted EETI loop 3 clones tested for binding to fluorescently-labeled trypsin are shown in their relative groups: (A) clones containing four or more common motifs, (B) clones containing three common motifs, and (C) clones least similar to those recovered from the enriched EL3-9 library. Names of the predicted clones are shown in bold. Numbers above the branch lines denote the number of other predicted clones in the same cluster as the selected clone. Percentages denote the average homology of the loop sequence of the predicted clone to those of the pool of enriched EL3-9 clones. The similarity rankings of each clone to the pool of enriched EL3-9 trypsin-binding clones, based on a modified BLOSUM62 substitution matrix, are shown in parentheses (where 1 is most similar and 420 is least similar).
    Figure Legend Snippet: Cladograms of predicted EETI loop 3 clones tested for their abilities to bind trypsin. Select predicted EETI loop 3 clones tested for binding to fluorescently-labeled trypsin are shown in their relative groups: (A) clones containing four or more common motifs, (B) clones containing three common motifs, and (C) clones least similar to those recovered from the enriched EL3-9 library. Names of the predicted clones are shown in bold. Numbers above the branch lines denote the number of other predicted clones in the same cluster as the selected clone. Percentages denote the average homology of the loop sequence of the predicted clone to those of the pool of enriched EL3-9 clones. The similarity rankings of each clone to the pool of enriched EL3-9 trypsin-binding clones, based on a modified BLOSUM62 substitution matrix, are shown in parentheses (where 1 is most similar and 420 is least similar).

    Techniques Used: Clone Assay, Binding Assay, Labeling, Sequencing, Modification

    Positional diversities and amino acid preferences of EETI loop 3-substituted clones. Positional diversities of the (A) EL3-6, (B) EL3-7, (C) EL3-8, and (D) EL3-9 libraries before sorting (grey) and after enriching for trypsin-binding clones (black) are depicted in the upper panels. A diversity score of 0.05 denotes complete conservation while a score of 1.0 signifies the presence of all 20 amino acids in equal proportions. Preferred amino acids at each loop-substituted position of enriched libraries are shown in the lower panels, with amino acids colored according to chemical property: polar (green), basic (blue), acidic (red), external polar (purple), and hydrophobic (black).
    Figure Legend Snippet: Positional diversities and amino acid preferences of EETI loop 3-substituted clones. Positional diversities of the (A) EL3-6, (B) EL3-7, (C) EL3-8, and (D) EL3-9 libraries before sorting (grey) and after enriching for trypsin-binding clones (black) are depicted in the upper panels. A diversity score of 0.05 denotes complete conservation while a score of 1.0 signifies the presence of all 20 amino acids in equal proportions. Preferred amino acids at each loop-substituted position of enriched libraries are shown in the lower panels, with amino acids colored according to chemical property: polar (green), basic (blue), acidic (red), external polar (purple), and hydrophobic (black).

    Techniques Used: Clone Assay, Binding Assay

    Schematic for interrogating the tolerance of sequence diversity in knottin loops. (A) Six libraries of loop-substituted knottin variants were designed based on the wild-type sequence of EETI. Libraries were created by replacing cysteine-flanked loop 2 (green) or loop 3 (blue) sequences with peptides of randomized amino acids (X) and varying lengths (n). The trypsin binding loop (orange) was not replaced, but instead used as a handle to evaluate the proper folding of EETI loop-substituted clones. Disulfide bonds are shown in yellow. (B) The binding interaction between trypsin (light grey) and EETI (PDB 2eti and 1h9h) is mediated through the trypsin binding loop, and is dependent on the correct formation of all three disulfide bonds. This interaction was exploited for high-throughput isolation of properly folded EETI loop-substituted variants.
    Figure Legend Snippet: Schematic for interrogating the tolerance of sequence diversity in knottin loops. (A) Six libraries of loop-substituted knottin variants were designed based on the wild-type sequence of EETI. Libraries were created by replacing cysteine-flanked loop 2 (green) or loop 3 (blue) sequences with peptides of randomized amino acids (X) and varying lengths (n). The trypsin binding loop (orange) was not replaced, but instead used as a handle to evaluate the proper folding of EETI loop-substituted clones. Disulfide bonds are shown in yellow. (B) The binding interaction between trypsin (light grey) and EETI (PDB 2eti and 1h9h) is mediated through the trypsin binding loop, and is dependent on the correct formation of all three disulfide bonds. This interaction was exploited for high-throughput isolation of properly folded EETI loop-substituted variants.

    Techniques Used: Sequencing, Binding Assay, Clone Assay, High Throughput Screening Assay, Isolation

    Covarying loop positions in EETI loop 3-substituted clones. Coupled loop positions in enriched EL3-9 clones are shown linked with their respective z-scores. Covariance patterns and correlated pairs of amino acids at each of the coupled positions were used to predict sequences of EETI loop 3 variants that adopt the knottin fold. For purposes of generating predictions, position three was set to asparagine or threonine, and positions eight and nine were set to glycine and tyrosine, respectively.
    Figure Legend Snippet: Covarying loop positions in EETI loop 3-substituted clones. Coupled loop positions in enriched EL3-9 clones are shown linked with their respective z-scores. Covariance patterns and correlated pairs of amino acids at each of the coupled positions were used to predict sequences of EETI loop 3 variants that adopt the knottin fold. For purposes of generating predictions, position three was set to asparagine or threonine, and positions eight and nine were set to glycine and tyrosine, respectively.

    Techniques Used: Clone Assay

    Sequence analysis of randomized peptides from EETI loop-substituted libraries. (A) Amino acid frequencies anticipated from an NNS degenerate codon loop library (black) are shown compared to the observed frequencies of randomized peptides from unsorted EETI loop 2-substituted (white) and EETI loop 3-substituted (grey) libraries. (B) The percent change of individual amino acid frequencies in the substituted loops of enriched EETI loop 2-substituted (white) and EETI loop 3-substituted (grey) clones compared to their frequencies in the respective unsorted libraries. (C) Amino acid frequencies in the randomized peptides of properly folded EETI loop 2-substituted (white) and EETI loop 3-substituted (grey) clones enriched for trypsin binding.
    Figure Legend Snippet: Sequence analysis of randomized peptides from EETI loop-substituted libraries. (A) Amino acid frequencies anticipated from an NNS degenerate codon loop library (black) are shown compared to the observed frequencies of randomized peptides from unsorted EETI loop 2-substituted (white) and EETI loop 3-substituted (grey) libraries. (B) The percent change of individual amino acid frequencies in the substituted loops of enriched EETI loop 2-substituted (white) and EETI loop 3-substituted (grey) clones compared to their frequencies in the respective unsorted libraries. (C) Amino acid frequencies in the randomized peptides of properly folded EETI loop 2-substituted (white) and EETI loop 3-substituted (grey) clones enriched for trypsin binding.

    Techniques Used: Sequencing, Binding Assay

    3) Product Images from "Horizontal gene transfer of a bacterial insect toxin gene into the Epichloë fungal symbionts of grasses"

    Article Title: Horizontal gene transfer of a bacterial insect toxin gene into the Epichloë fungal symbionts of grasses

    Journal: Scientific Reports

    doi: 10.1038/srep05562

    Gene structure of the Epichloë mcf-like genes and amino acid similarity of E. typhina s ubsp. poae Ps1-Mcf protein with the bacterial Mcf proteins. (a) Diagrams of gene structure of the bacterial mcf1 , mcf2 , fitD , and Epichloë mcf-like genes. The exons are indicated by boxes and the introns by lines. The conserved TcdA/TcdB pore-forming region is indicated by filled in boxes. The positions of the premature stop codons in some of the Epichloë genes are indicated by *. The end of intron 1 of the E. typhina s ubsp. poae 5819 sequence was modified from the annotated version to that of the experimentally determined position in E. typhina s ubsp. poae Ps1. (b) GECA analysis illustrating the amino acid sequence similarity of the E. typhina s ubsp. poae Ps1-Mcf protein (1,997 amino acids; accession KJ502561) to Mcf1 (2,929 amino acids; accession AF503504.2) and Mcf2 (2,388 amino acids; accession AY445665) proteins from Ph. luminescens.
    Figure Legend Snippet: Gene structure of the Epichloë mcf-like genes and amino acid similarity of E. typhina s ubsp. poae Ps1-Mcf protein with the bacterial Mcf proteins. (a) Diagrams of gene structure of the bacterial mcf1 , mcf2 , fitD , and Epichloë mcf-like genes. The exons are indicated by boxes and the introns by lines. The conserved TcdA/TcdB pore-forming region is indicated by filled in boxes. The positions of the premature stop codons in some of the Epichloë genes are indicated by *. The end of intron 1 of the E. typhina s ubsp. poae 5819 sequence was modified from the annotated version to that of the experimentally determined position in E. typhina s ubsp. poae Ps1. (b) GECA analysis illustrating the amino acid sequence similarity of the E. typhina s ubsp. poae Ps1-Mcf protein (1,997 amino acids; accession KJ502561) to Mcf1 (2,929 amino acids; accession AF503504.2) and Mcf2 (2,388 amino acids; accession AY445665) proteins from Ph. luminescens.

    Techniques Used: Sequencing, Modification

    4) Product Images from "Most commonly mutated genes in High Grade Serous Ovarian Carcinoma are nonessential for ovarian surface epithelial stem cell transformation"

    Article Title: Most commonly mutated genes in High Grade Serous Ovarian Carcinoma are nonessential for ovarian surface epithelial stem cell transformation

    Journal: bioRxiv

    doi: 10.1101/2020.01.27.921718

    OSE-SC (ALDH+) transform more frequently than OSE-NS (ALDH-) despite similar viral transduction rates. (A) Percent transformation of OSE-SC, OSE-NS and unsorted OSE following LentiCRISPRv2 minilibrary transduction. OSE-SC transformed more frequently than OSE-NS and unsorted OSE. Unsorted OSE transformed more frequently than OSE-NS (5 replicates. SEM error bars). ( B,C) FUGW-mCherry (mCherry-expressing lentivirus) transduction efficiency in OSE-SC and OSE-NS detected via flow cytometry. Flow cytometry was used to count mCherry+ cells following transduction with equal concentrations of FUGW-mCherry lentivirus. Percentages indicate the percentage of total cells that are mCherry+. The dark grey lines represent cell counts of untransduced cells. The red line represents cell counts of FUGW-mCherry transduced cells. OSE-SC and OSE-NS gained mCherry fluorescence at similar rates following lentiviral transduction.
    Figure Legend Snippet: OSE-SC (ALDH+) transform more frequently than OSE-NS (ALDH-) despite similar viral transduction rates. (A) Percent transformation of OSE-SC, OSE-NS and unsorted OSE following LentiCRISPRv2 minilibrary transduction. OSE-SC transformed more frequently than OSE-NS and unsorted OSE. Unsorted OSE transformed more frequently than OSE-NS (5 replicates. SEM error bars). ( B,C) FUGW-mCherry (mCherry-expressing lentivirus) transduction efficiency in OSE-SC and OSE-NS detected via flow cytometry. Flow cytometry was used to count mCherry+ cells following transduction with equal concentrations of FUGW-mCherry lentivirus. Percentages indicate the percentage of total cells that are mCherry+. The dark grey lines represent cell counts of untransduced cells. The red line represents cell counts of FUGW-mCherry transduced cells. OSE-SC and OSE-NS gained mCherry fluorescence at similar rates following lentiviral transduction.

    Techniques Used: Transduction, Transformation Assay, Expressing, Flow Cytometry, Fluorescence

    Identification of genome-integrated LentiCRISPRs overrepresented target gene combinations in OSE-NS. (A) Percent gene targeting frequency in OSE-NS colonies. (B) Genome-integration and hierarchical clustering of LentiCRISPRv2 constructs in OSE-SC samples. The binary color scale shows whether a gene is targeted by at least one lentiCRISPR in each individual sample. Light grey indicates that a given gene was not targeted, while dark grey indicates that a gene was targeted by at least one LentiCRISPRv2 construct. Hierarchical clustering was performed on both sample similarity and gene targeting, resulting in several clusters of co-targeted genes and similar transformants.
    Figure Legend Snippet: Identification of genome-integrated LentiCRISPRs overrepresented target gene combinations in OSE-NS. (A) Percent gene targeting frequency in OSE-NS colonies. (B) Genome-integration and hierarchical clustering of LentiCRISPRv2 constructs in OSE-SC samples. The binary color scale shows whether a gene is targeted by at least one lentiCRISPR in each individual sample. Light grey indicates that a given gene was not targeted, while dark grey indicates that a gene was targeted by at least one LentiCRISPRv2 construct. Hierarchical clustering was performed on both sample similarity and gene targeting, resulting in several clusters of co-targeted genes and similar transformants.

    Techniques Used: Construct

    Identification of genome-integrated LentiCRISPRs and overrepresented target gene combinations in OSE-SC. (A) Genome-integration and hierarchical clustering of LentiCRISPRv2 constructs in OSE-SC samples. Hierarchical clustering was performed on both sample similarity and gene targeting. (B) Overall percent gene targeting and co-targeting frequency. Significance for single integration overall was assessed using Chi2 (df=19) and is indicated with an asterisk. The heat map displays co-integration frequency of each gene present on the x axis with a gene shown on the y axis. (C) Overrepresentation of co-targeted genes in sample subgroups. Over or underrepresentation was determined using Chi2 analyses. Chi2 values corresponding to p ≤ 0.05 (df = 19) are colored in green. Red coloration indicates that co-integration may have occurred by chance, and that the p value is ≥ 0.05.
    Figure Legend Snippet: Identification of genome-integrated LentiCRISPRs and overrepresented target gene combinations in OSE-SC. (A) Genome-integration and hierarchical clustering of LentiCRISPRv2 constructs in OSE-SC samples. Hierarchical clustering was performed on both sample similarity and gene targeting. (B) Overall percent gene targeting and co-targeting frequency. Significance for single integration overall was assessed using Chi2 (df=19) and is indicated with an asterisk. The heat map displays co-integration frequency of each gene present on the x axis with a gene shown on the y axis. (C) Overrepresentation of co-targeted genes in sample subgroups. Over or underrepresentation was determined using Chi2 analyses. Chi2 values corresponding to p ≤ 0.05 (df = 19) are colored in green. Red coloration indicates that co-integration may have occurred by chance, and that the p value is ≥ 0.05.

    Techniques Used: Construct

    Targeted OSE-SC-transformation assay and validation of overrepresented LentiCRISPR combinations. A baseline level of adhesion independent growth was first assessed via induction of specific “core mutations” via LentiCRISPRv2 targeting. Additional minilibrary target genes were then mutated (using LentiCRISPRv2) alongside core mutations to assess whether they act synergistically to promote adhesion independent growth. Colony counts that are significantly greater than baseline rates (core mutations alone) are labeled with an asterisk (*) (Students’ two-tailed t-test p
    Figure Legend Snippet: Targeted OSE-SC-transformation assay and validation of overrepresented LentiCRISPR combinations. A baseline level of adhesion independent growth was first assessed via induction of specific “core mutations” via LentiCRISPRv2 targeting. Additional minilibrary target genes were then mutated (using LentiCRISPRv2) alongside core mutations to assess whether they act synergistically to promote adhesion independent growth. Colony counts that are significantly greater than baseline rates (core mutations alone) are labeled with an asterisk (*) (Students’ two-tailed t-test p

    Techniques Used: Transformation Assay, Labeling, Two Tailed Test

    Strategy for identifying HGSOC tumor suppressor combinations. A total of 60 constructs were made in the vector LentiCRISPRv2, constituting the “minilibrary”. OSE-NS or OSE-SC were transduced with functionally-validated LentiCRISPRs and then plated in soft agar. Individual transformants/colonies were isolated and individually cultured. Genome-integrated LentiCRISPRs from each transformant were identified by sequencing, and overrepresented combinations later validated in directed soft agar transformation assays. OSE-SC, ovarian surface epithelium stem cells; OSE-NS, ovarian surface epithelium non-stem cells; NGS, next-generation sequencing.
    Figure Legend Snippet: Strategy for identifying HGSOC tumor suppressor combinations. A total of 60 constructs were made in the vector LentiCRISPRv2, constituting the “minilibrary”. OSE-NS or OSE-SC were transduced with functionally-validated LentiCRISPRs and then plated in soft agar. Individual transformants/colonies were isolated and individually cultured. Genome-integrated LentiCRISPRs from each transformant were identified by sequencing, and overrepresented combinations later validated in directed soft agar transformation assays. OSE-SC, ovarian surface epithelium stem cells; OSE-NS, ovarian surface epithelium non-stem cells; NGS, next-generation sequencing.

    Techniques Used: Construct, Plasmid Preparation, Transduction, Isolation, Cell Culture, Sequencing, Transformation Assay, Next-Generation Sequencing

    5) Product Images from "A Novel pH-Regulated, Unusual 603 bp Overlapping Protein Coding Gene pop Is Encoded Antisense to ompA in Escherichia coli O157:H7 (EHEC)"

    Article Title: A Novel pH-Regulated, Unusual 603 bp Overlapping Protein Coding Gene pop Is Encoded Antisense to ompA in Escherichia coli O157:H7 (EHEC)

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2020.00377

    Inter-strain comparison of pop translation. Alignment of sequence and ribosomal profiling reads of pop and its homologs across pathogenic E. coli strains O157:H7 EDL933 (A) , O157:H7 Sakai (B) , and LF82 (C) . Graphs show normalized sequencing reads (RPKM, reads per kilobase per million mapped reads) of ribosome profiling experiments in LB medium (A,B) or Schaedler broth (C) ; the sum signal of two biological replicates is visualized. Three putative start codons are indicated with green dashed lines in region 1 (TTG), 2 (CTG), and 3 (GTG). The stop codon is indicated by a red dashed line. (D) Averaged RPKM values of translation and (E) ribosomal coverage values (RCV) of overlapping gene pop and the upstream annotated gene ycbG of three E. coli strains. Purple dashed line in panel (E) : threshold for translated ORFs, RCV = 0.35. Error bars indicate both underlying values used for calculations.
    Figure Legend Snippet: Inter-strain comparison of pop translation. Alignment of sequence and ribosomal profiling reads of pop and its homologs across pathogenic E. coli strains O157:H7 EDL933 (A) , O157:H7 Sakai (B) , and LF82 (C) . Graphs show normalized sequencing reads (RPKM, reads per kilobase per million mapped reads) of ribosome profiling experiments in LB medium (A,B) or Schaedler broth (C) ; the sum signal of two biological replicates is visualized. Three putative start codons are indicated with green dashed lines in region 1 (TTG), 2 (CTG), and 3 (GTG). The stop codon is indicated by a red dashed line. (D) Averaged RPKM values of translation and (E) ribosomal coverage values (RCV) of overlapping gene pop and the upstream annotated gene ycbG of three E. coli strains. Purple dashed line in panel (E) : threshold for translated ORFs, RCV = 0.35. Error bars indicate both underlying values used for calculations.

    Techniques Used: Sequencing

    6) Product Images from "Physiologic RNA Targets and Refined Sequence Specificity of Coronavirus EndoU"

    Article Title: Physiologic RNA Targets and Refined Sequence Specificity of Coronavirus EndoU

    Journal: bioRxiv

    doi: 10.1101/2020.05.20.064436

    Frequency of endoribonuclease cleavage in host and viral RNAs. (A and B) Normalized cyclic phosphate cDNA reads ([reads at each position / total reads in library]) mapped to host and viral RNAs at 9 and 12 hpi in WT, IFNAR -/- , and RNase L -/- bone marrow macrophages (BMM).
    Figure Legend Snippet: Frequency of endoribonuclease cleavage in host and viral RNAs. (A and B) Normalized cyclic phosphate cDNA reads ([reads at each position / total reads in library]) mapped to host and viral RNAs at 9 and 12 hpi in WT, IFNAR -/- , and RNase L -/- bone marrow macrophages (BMM).

    Techniques Used:

    MHV secondary structures associated with RNase L-dependent and EndoU-dependent cleavage sites. (A and C) Nucleotide resolution graphs displaying normalized counts by position for the regions encompassing secondary structure predictions. (B and D) Secondary structures of frameshift stimulation element (B) and MHV 3’-UTR pseudoknot (D), generated using available consensus alignment and the R-scape program ( 85 ). MHV A59 sequence mapped to consensus secondary structures using available covariation model and the Infernal program ( 86 ). Base coloring of MHV A59 sequence based on normalized cDNA reads as indicated in key for 12 hpi in WT BMM infected with MHV (V) . *Base RNase L-dependent cleavage activity is increased in PDE mut or EndoU mut infection as compared to MHV (V) infection.
    Figure Legend Snippet: MHV secondary structures associated with RNase L-dependent and EndoU-dependent cleavage sites. (A and C) Nucleotide resolution graphs displaying normalized counts by position for the regions encompassing secondary structure predictions. (B and D) Secondary structures of frameshift stimulation element (B) and MHV 3’-UTR pseudoknot (D), generated using available consensus alignment and the R-scape program ( 85 ). MHV A59 sequence mapped to consensus secondary structures using available covariation model and the Infernal program ( 86 ). Base coloring of MHV A59 sequence based on normalized cDNA reads as indicated in key for 12 hpi in WT BMM infected with MHV (V) . *Base RNase L-dependent cleavage activity is increased in PDE mut or EndoU mut infection as compared to MHV (V) infection.

    Techniques Used: Generated, Sequencing, Infection, Activity Assay

    Frequency and location of endoribonuclease cleavage sites in MHV genomic RNA. (A and B) Normalized cyclic phosphate cDNA reads captured at each position along the MHV genomic RNA at 9 and 12 hpi with MHV (S), MHV (V) , PDE mut , and EndoU mut virus in (A) WT BMM, (B) IFNAR -/- , and (C) RNase L -/- BMM. Putative cleavage sites attributed to EndoU or RNase L were calculated from RNase L- or EndoU-dependent signal generated by subtracting signal from each captured position that occurs in the absence of either enzyme (RNase L -/- BMM or during EndoU mut infection). These data were then filtered for sites with reads representing at least 0.01 % of total reads in the library. At each of these positions, the log 2 fold change in signal when either RNase L or EndoU were absent was calculated and sites with ≥ 2.5 fold change were designated putative RNase L or EndoU sites.
    Figure Legend Snippet: Frequency and location of endoribonuclease cleavage sites in MHV genomic RNA. (A and B) Normalized cyclic phosphate cDNA reads captured at each position along the MHV genomic RNA at 9 and 12 hpi with MHV (S), MHV (V) , PDE mut , and EndoU mut virus in (A) WT BMM, (B) IFNAR -/- , and (C) RNase L -/- BMM. Putative cleavage sites attributed to EndoU or RNase L were calculated from RNase L- or EndoU-dependent signal generated by subtracting signal from each captured position that occurs in the absence of either enzyme (RNase L -/- BMM or during EndoU mut infection). These data were then filtered for sites with reads representing at least 0.01 % of total reads in the library. At each of these positions, the log 2 fold change in signal when either RNase L or EndoU were absent was calculated and sites with ≥ 2.5 fold change were designated putative RNase L or EndoU sites.

    Techniques Used: Generated, Infection

    Abundance of cyclic phosphate ends by MHV genomic region and MHV mRNA abundance. Sum of endonuclease cleavage sites in MHV RNA, by genomic regions: sum of cyclic phosphate reads (A), sum of cyclic phosphate reads normalized by MHV mRNA abundance (B) or sum of cyclic phosphate reads normalized by the length of the MHV genomic region (C). (A) Sum of cyclic phosphate cDNA reads displayed by MHV RNA region for WT, IFNAR -/- , RNase L -/- BMM across all conditions of viral infection at 12 hpi. Transcriptional regulatory sequences (TRS) are numbered by their associated mRNA ( 2 – 7 ). Other MHV genomic regions are labeled as shown in Figure 1A . (B) Frequency of endonuclease cleavage sites in MHV RNA, by genomic regions, normalized by MHV mRNA abundance. Sum of cyclic phosphate counts normalized by mRNA abundance at each capture base, displayed by MHV genomic region for WT, IFNAR -/- , RNase L -/- BMM across all conditions of viral infection at 12 hpi. (C) Percent of sum of normalized counts per length of MHV genomic region for WT, IFNAR -/- and RNase L -/- BMM across all conditions of viral infection at 12 hpi. Dotted line represents baseline percent of cleavage expected by cell type ([total number of cyclic phosphate counts / total genome size x 100]). (D) Frequency and location of cleavage in the MHV TRS elements in WT BMM during infection with MHV (V) and EndoU mut at 12 hpi. The x-axis includes the sequence and position of the 6-base MHV TRS elements. (E) Normalized counts (sum of MHV sg mRNA / sum of all MHV mRNAs) of MHV sg mRNAs detected in WT, IFNAR -/- , RNase L -/- BMM across all conditions of viral infection at 9 and 12 hpi. (F) Sum of all MHV sg mRNAs (RPM) for WT, IFNAR -/- , RNase L -/- BMM across all conditions of viral infection at 9 and 12 hpi.
    Figure Legend Snippet: Abundance of cyclic phosphate ends by MHV genomic region and MHV mRNA abundance. Sum of endonuclease cleavage sites in MHV RNA, by genomic regions: sum of cyclic phosphate reads (A), sum of cyclic phosphate reads normalized by MHV mRNA abundance (B) or sum of cyclic phosphate reads normalized by the length of the MHV genomic region (C). (A) Sum of cyclic phosphate cDNA reads displayed by MHV RNA region for WT, IFNAR -/- , RNase L -/- BMM across all conditions of viral infection at 12 hpi. Transcriptional regulatory sequences (TRS) are numbered by their associated mRNA ( 2 – 7 ). Other MHV genomic regions are labeled as shown in Figure 1A . (B) Frequency of endonuclease cleavage sites in MHV RNA, by genomic regions, normalized by MHV mRNA abundance. Sum of cyclic phosphate counts normalized by mRNA abundance at each capture base, displayed by MHV genomic region for WT, IFNAR -/- , RNase L -/- BMM across all conditions of viral infection at 12 hpi. (C) Percent of sum of normalized counts per length of MHV genomic region for WT, IFNAR -/- and RNase L -/- BMM across all conditions of viral infection at 12 hpi. Dotted line represents baseline percent of cleavage expected by cell type ([total number of cyclic phosphate counts / total genome size x 100]). (D) Frequency and location of cleavage in the MHV TRS elements in WT BMM during infection with MHV (V) and EndoU mut at 12 hpi. The x-axis includes the sequence and position of the 6-base MHV TRS elements. (E) Normalized counts (sum of MHV sg mRNA / sum of all MHV mRNAs) of MHV sg mRNAs detected in WT, IFNAR -/- , RNase L -/- BMM across all conditions of viral infection at 9 and 12 hpi. (F) Sum of all MHV sg mRNAs (RPM) for WT, IFNAR -/- , RNase L -/- BMM across all conditions of viral infection at 9 and 12 hpi.

    Techniques Used: Infection, Labeling, Sequencing

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

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

    Journal: bioRxiv

    doi: 10.1101/852251

    Inter-strain comparison of pop translation. Alignment of sequence and ribosomal profiling reads of pop and its homologs across pathogenic E. coli strains O157:H7 EDL933 (A) , O157:H7 Sakai (B) , and LF82 (C) . Graphs show normalized sequencing reads (RPKM, reads per kilobase per million mapped reads) of ribosome profiling experiments in LB medium (A, B) or Schaedler broth (C) ; the sum signal of two biological replicates is visualized. Three putative start codons are indicated with green dashed lines in region 1 (TTG), 2 (CTG) and 3 (GTG). The stop codon is indicated by a red dashed line. (D) Averaged RPKM values of translation and (E) ribosomal coverage values (RCV) of overlapping gene pop and the upstream annotated gene ycbG of three pathogenic strains. Purple dashed line in (E): threshold for translated ORFs, RCV=0.355.
    Figure Legend Snippet: Inter-strain comparison of pop translation. Alignment of sequence and ribosomal profiling reads of pop and its homologs across pathogenic E. coli strains O157:H7 EDL933 (A) , O157:H7 Sakai (B) , and LF82 (C) . Graphs show normalized sequencing reads (RPKM, reads per kilobase per million mapped reads) of ribosome profiling experiments in LB medium (A, B) or Schaedler broth (C) ; the sum signal of two biological replicates is visualized. Three putative start codons are indicated with green dashed lines in region 1 (TTG), 2 (CTG) and 3 (GTG). The stop codon is indicated by a red dashed line. (D) Averaged RPKM values of translation and (E) ribosomal coverage values (RCV) of overlapping gene pop and the upstream annotated gene ycbG of three pathogenic strains. Purple dashed line in (E): threshold for translated ORFs, RCV=0.355.

    Techniques Used: Sequencing

    8) Product Images from "Abrogation of PRRSV infectivity by CRISPR-Cas13b-mediated viral RNA cleavage in mammalian cells"

    Article Title: Abrogation of PRRSV infectivity by CRISPR-Cas13b-mediated viral RNA cleavage in mammalian cells

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-66775-3

    Development of an all-in-one CRISPR/Cas13b-duplexed crRNA delivery system. ( a ) The structure of the all-in-one delivery vector and PRRSV gene reporter. The U6 promoter drives duplexed guide expression, and the EF1a promoter mediates the transcription of Cas13b. The eGFP reporter fused with Cas13b by a 2 A self-cleaving peptide facilitates the detection of Cas13b expression. ( b , c , d ) The all-in-one Cas13b system carrying specific PRRSV ORF5 crRNA significantly cleaved ORF5 mRNA. However, no accumulative effect of Cas13b was detected by targeting the ORF5 gene with two crRNAs simultaneously ( b , c ), flow cytometry; ( d) , real-time PCR). ( e ) The all-in-one plasmid was further modified to incorporate triple crRNAs targeting the ORF7 gene. Representative flow cytometry graphs for eGFP- and RFP657-positive cells are shown. ( f ) The reduction percentages of dual-marker positive cells were determined by flow cytometry. Each crRNA group was normalized to the NT control. ( g ) The percentage of RNA reduction was determined by qRT-PCR. ( h ) The established platform enables the simultaneous knockdown of PRRSV ORF5 and ORF7 mRNA by incorporating two corresponding guide protospacer sequences. Values are shown as mean ± SEM with n = 3. **, ***and NS refer to P values
    Figure Legend Snippet: Development of an all-in-one CRISPR/Cas13b-duplexed crRNA delivery system. ( a ) The structure of the all-in-one delivery vector and PRRSV gene reporter. The U6 promoter drives duplexed guide expression, and the EF1a promoter mediates the transcription of Cas13b. The eGFP reporter fused with Cas13b by a 2 A self-cleaving peptide facilitates the detection of Cas13b expression. ( b , c , d ) The all-in-one Cas13b system carrying specific PRRSV ORF5 crRNA significantly cleaved ORF5 mRNA. However, no accumulative effect of Cas13b was detected by targeting the ORF5 gene with two crRNAs simultaneously ( b , c ), flow cytometry; ( d) , real-time PCR). ( e ) The all-in-one plasmid was further modified to incorporate triple crRNAs targeting the ORF7 gene. Representative flow cytometry graphs for eGFP- and RFP657-positive cells are shown. ( f ) The reduction percentages of dual-marker positive cells were determined by flow cytometry. Each crRNA group was normalized to the NT control. ( g ) The percentage of RNA reduction was determined by qRT-PCR. ( h ) The established platform enables the simultaneous knockdown of PRRSV ORF5 and ORF7 mRNA by incorporating two corresponding guide protospacer sequences. Values are shown as mean ± SEM with n = 3. **, ***and NS refer to P values

    Techniques Used: CRISPR, Plasmid Preparation, Expressing, Flow Cytometry, Real-time Polymerase Chain Reaction, Modification, Marker, Quantitative RT-PCR

    Cas13b mediates the efficient knockdown of the PRRSV genome in lentiviral transgenic MARC-145 cells. ( a ) Schematic diagram for lentiviral transfer gene constructs encoding Cas13b and crRNAs. ( b ) Determination of Cas13 effector expression levels in puro-selected transgenic cells by flow cytometry analysis. ( c ) The expression of corresponding crRNAs in each cell line was detected by PCR. PCR products were separated by 5% agarose gels. ( d ) The growth kinetics of HP-PRRSV strain 10PL01 with an MOI of 0.1 in transgenic cells. ( e ) The Cas13b cleavage activity on PRRSV genomic RNA was determined by qRT-PCR with primers targeting the NSP9 gene. ( f ) The PRRSV subgenomic RNA levels were measured by qRT-PCR with a set of specific primers targeting each subgenomic RNA. ( g ) Comparison of each subgenomic RNA knockdown efficiency between cells expressing Cas13b-crRNA 5-2 and Cas13b-crRNA 7-1. Values are shown as the mean ± SEM with n = 3. ***refers to P value
    Figure Legend Snippet: Cas13b mediates the efficient knockdown of the PRRSV genome in lentiviral transgenic MARC-145 cells. ( a ) Schematic diagram for lentiviral transfer gene constructs encoding Cas13b and crRNAs. ( b ) Determination of Cas13 effector expression levels in puro-selected transgenic cells by flow cytometry analysis. ( c ) The expression of corresponding crRNAs in each cell line was detected by PCR. PCR products were separated by 5% agarose gels. ( d ) The growth kinetics of HP-PRRSV strain 10PL01 with an MOI of 0.1 in transgenic cells. ( e ) The Cas13b cleavage activity on PRRSV genomic RNA was determined by qRT-PCR with primers targeting the NSP9 gene. ( f ) The PRRSV subgenomic RNA levels were measured by qRT-PCR with a set of specific primers targeting each subgenomic RNA. ( g ) Comparison of each subgenomic RNA knockdown efficiency between cells expressing Cas13b-crRNA 5-2 and Cas13b-crRNA 7-1. Values are shown as the mean ± SEM with n = 3. ***refers to P value

    Techniques Used: Transgenic Assay, Construct, Expressing, Flow Cytometry, Polymerase Chain Reaction, Activity Assay, Quantitative RT-PCR

    Characterization of the CRISPR/Cas13b system in PRRSV mRNA targeting. ( a ) Schematic diagram of the design of crRNAs targeting the ORF7 mRNA transcript, template strand of the ORF7 gene and CMV promoter. ( b ) Schematic diagram showing the steps of the determination of the effect of CRISPR/Cas13b on PRRSV gene knockdown by co-transfection of the three plasmids into HEK293T cells. ( c ) Microscopic fluorescence images showing the expression of the PRRSV ORF7-eGFP reporter after CRISPR/Cas13b activity with various targeting crRNAs. The bar indicates 100 μm. ( d , e ) PRRSV N protein expression and ORF7 mRNA levels were determined by flow cytometry and quantitative RT-PCR, respectively. Values shown as the mean ± SEM with n = 3. *and **refer to P values
    Figure Legend Snippet: Characterization of the CRISPR/Cas13b system in PRRSV mRNA targeting. ( a ) Schematic diagram of the design of crRNAs targeting the ORF7 mRNA transcript, template strand of the ORF7 gene and CMV promoter. ( b ) Schematic diagram showing the steps of the determination of the effect of CRISPR/Cas13b on PRRSV gene knockdown by co-transfection of the three plasmids into HEK293T cells. ( c ) Microscopic fluorescence images showing the expression of the PRRSV ORF7-eGFP reporter after CRISPR/Cas13b activity with various targeting crRNAs. The bar indicates 100 μm. ( d , e ) PRRSV N protein expression and ORF7 mRNA levels were determined by flow cytometry and quantitative RT-PCR, respectively. Values shown as the mean ± SEM with n = 3. *and **refer to P values

    Techniques Used: CRISPR, Cotransfection, Fluorescence, Expressing, Activity Assay, Flow Cytometry, Quantitative RT-PCR

    Determination of the most potent crRNA for Cas13b-mediated PRRSV ORF5 and ORF7 targeting. ( a ) Locations of crRNA targeting regions within the ORF5 and ORF7 genes. ( b ) RNA cleavage efficiency was determined for the indicated crRNAs targeting ORF5 and ORF7 by qRT-PCR and normalized to the non-targeting (NT) control.
    Figure Legend Snippet: Determination of the most potent crRNA for Cas13b-mediated PRRSV ORF5 and ORF7 targeting. ( a ) Locations of crRNA targeting regions within the ORF5 and ORF7 genes. ( b ) RNA cleavage efficiency was determined for the indicated crRNAs targeting ORF5 and ORF7 by qRT-PCR and normalized to the non-targeting (NT) control.

    Techniques Used: Quantitative RT-PCR

    9) Product Images from "Functional validation of GPIHBP1 and identification of a functional mutation in GPIHBP1 for milk fat traits in dairy cattle"

    Article Title: Functional validation of GPIHBP1 and identification of a functional mutation in GPIHBP1 for milk fat traits in dairy cattle

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-08668-6

    Promoter activity analysis of the bovine GPIHBP1 gene. There are four completely linked SNPs in the promoter region of GPIHBP1 . As shown, fragments P1, P2, P3, and P4 contain one, two, three, and four SNPs sites, respectively. We constructed eight types of recombinant promoter vectors with respect to the four fragments and the mutant and wild-type haplotypes. Promoter activities were detected using a dual-luciferase reporter system. The data are expressed as the means and standard errors of three replicates.
    Figure Legend Snippet: Promoter activity analysis of the bovine GPIHBP1 gene. There are four completely linked SNPs in the promoter region of GPIHBP1 . As shown, fragments P1, P2, P3, and P4 contain one, two, three, and four SNPs sites, respectively. We constructed eight types of recombinant promoter vectors with respect to the four fragments and the mutant and wild-type haplotypes. Promoter activities were detected using a dual-luciferase reporter system. The data are expressed as the means and standard errors of three replicates.

    Techniques Used: Activity Assay, Construct, Recombinant, Mutagenesis, Luciferase

    Expression of GPIHBP1 and five milk fat-related genes in bovine primary mammary epithelial cells that were transfected with the pcDNA3.1(+)-GPIHBP1 eukaryotic expression vector (over-expression) and with an empty vector pcDNA 3.1(+) (control). The vertical axes represent the expression of these genes relative to the expression of the housekeeping gene GAPDH .
    Figure Legend Snippet: Expression of GPIHBP1 and five milk fat-related genes in bovine primary mammary epithelial cells that were transfected with the pcDNA3.1(+)-GPIHBP1 eukaryotic expression vector (over-expression) and with an empty vector pcDNA 3.1(+) (control). The vertical axes represent the expression of these genes relative to the expression of the housekeeping gene GAPDH .

    Techniques Used: Expressing, Transfection, Plasmid Preparation, Over Expression

    Expression of GPIHBP1 in the mammary gland of cows with genotypes AA, GG, and GA.
    Figure Legend Snippet: Expression of GPIHBP1 in the mammary gland of cows with genotypes AA, GG, and GA.

    Techniques Used: Expressing

    Expression of GPIHBP1 and five milk fat-related genes in bovine primary mammary epithelial cells that were transfected with Stealth™ RNAi siRNA targeting the bovine GPIHBP1 gene open reading frame (siRNA) and with the Stealth™ RNAi siRNA Negative Control (control). The vertical axes represent the expression of these genes relative to the expression of the housekeeping gene GAPDH .
    Figure Legend Snippet: Expression of GPIHBP1 and five milk fat-related genes in bovine primary mammary epithelial cells that were transfected with Stealth™ RNAi siRNA targeting the bovine GPIHBP1 gene open reading frame (siRNA) and with the Stealth™ RNAi siRNA Negative Control (control). The vertical axes represent the expression of these genes relative to the expression of the housekeeping gene GAPDH .

    Techniques Used: Expressing, Transfection, Negative Control

    Relative promoter activities of GPIHBP1 of four different promoter vectors with respect to the four haplotypes (GCGA, ACGA, GAAG, and AAAG). Promoter activities were detected using a dual-luciferase reporter system. The data are expressed as the means and standard errors of three replicates ** P
    Figure Legend Snippet: Relative promoter activities of GPIHBP1 of four different promoter vectors with respect to the four haplotypes (GCGA, ACGA, GAAG, and AAAG). Promoter activities were detected using a dual-luciferase reporter system. The data are expressed as the means and standard errors of three replicates ** P

    Techniques Used: Luciferase

    PCR amplification of the open reading frame (ORF) and different promoter segments of GPIHBP1 . The DNA markers were DL2000:2000bp, 1000 bp, 750 bp, 500 bp, 250 bp, 100 bp. ( A ) Band of the ORF (size = 544 bp). ( B ) Band of the promoter fragment (size = 2177 bp) that contains all four SNPs. ( C ) Band of the promoter fragment (size = 1646bp) that contains the first three SNPs. ( D ) Band of the promoter fragment (size = 1413 bp) that contains the first two SNPs. ( E ) Band of the promoter fragment (size = 482 bp) that contains only the first SNP.
    Figure Legend Snippet: PCR amplification of the open reading frame (ORF) and different promoter segments of GPIHBP1 . The DNA markers were DL2000:2000bp, 1000 bp, 750 bp, 500 bp, 250 bp, 100 bp. ( A ) Band of the ORF (size = 544 bp). ( B ) Band of the promoter fragment (size = 2177 bp) that contains all four SNPs. ( C ) Band of the promoter fragment (size = 1646bp) that contains the first three SNPs. ( D ) Band of the promoter fragment (size = 1413 bp) that contains the first two SNPs. ( E ) Band of the promoter fragment (size = 482 bp) that contains only the first SNP.

    Techniques Used: Polymerase Chain Reaction, Amplification

    10) Product Images from "DNA polymerase β uses its lyase domain in a processive search for DNA damage"

    Article Title: DNA polymerase β uses its lyase domain in a processive search for DNA damage

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx047

    Models of Pol β DNA damage location. ( A ) DNA damage (i.e. 1-nt gaps) are shown as black circles. Model 1 depicts facilitated diffusion which involves Pol β using DNA as a conduit to locate damage. Although depicted as directional for brevity, facilitated diffusion is stochastic. Model 2 represents 3D diffusion, where Pol β damage location depends on random and direct collisions with substrate. Pol β recruitment by protein-protein interactions is represented in model 3. ( B ) Three modes of facilitated diffusion.
    Figure Legend Snippet: Models of Pol β DNA damage location. ( A ) DNA damage (i.e. 1-nt gaps) are shown as black circles. Model 1 depicts facilitated diffusion which involves Pol β using DNA as a conduit to locate damage. Although depicted as directional for brevity, facilitated diffusion is stochastic. Model 2 represents 3D diffusion, where Pol β damage location depends on random and direct collisions with substrate. Pol β recruitment by protein-protein interactions is represented in model 3. ( B ) Three modes of facilitated diffusion.

    Techniques Used: Diffusion-based Assay

    Pol β searches DNA using a hopping mechanism. ( A ) The F p values for substrates containing 1-nt gaps on opposing strands (P20-OP) and with a small molecule roadblock (P20-RB) are compared to a substrate with 1-nt gaps on the same strand (P20). ( B ) The F p values were measured at 100 mM and 200 mM ionic strengths. The mean and standard deviation is reported from three independent experiments.
    Figure Legend Snippet: Pol β searches DNA using a hopping mechanism. ( A ) The F p values for substrates containing 1-nt gaps on opposing strands (P20-OP) and with a small molecule roadblock (P20-RB) are compared to a substrate with 1-nt gaps on the same strand (P20). ( B ) The F p values were measured at 100 mM and 200 mM ionic strengths. The mean and standard deviation is reported from three independent experiments.

    Techniques Used: Standard Deviation

    Site-spacing dependence of F p normalized by the insertion efficiency ( E ). The F p was measured for processive substrates containing 10, 20, 40 and 80 bp spacing distances (bottom x-axis) at 150 mM ionic strength. The top x-axis represents the distance (nm) between the two 1-nt gaps. The mean and standard deviation from three independent experiments is shown. The F p is normalized by the efficiency of insertion ( Supplementary Figure S4 ). The red line represents the best fit to a sliding mechanism (Equation 5 ). The blue line is a fit to a hopping mechanism (Equation 6 ).
    Figure Legend Snippet: Site-spacing dependence of F p normalized by the insertion efficiency ( E ). The F p was measured for processive substrates containing 10, 20, 40 and 80 bp spacing distances (bottom x-axis) at 150 mM ionic strength. The top x-axis represents the distance (nm) between the two 1-nt gaps. The mean and standard deviation from three independent experiments is shown. The F p is normalized by the efficiency of insertion ( Supplementary Figure S4 ). The red line represents the best fit to a sliding mechanism (Equation 5 ). The blue line is a fit to a hopping mechanism (Equation 6 ).

    Techniques Used: Standard Deviation

    Pol β catalyzed nucleotide insertion is reduced in the 601 NCP resulting in a lack of processivity. ( A ) NCP processive substrate design. Schematic depicting positions of 1-nt gaps and labeling strategy. Oligonucleotide B is 5΄ labeled with a 32 P phosphate. Site one is located 15 nts and site two 32 nts from the 5΄ end. Reactions were performed as described in Figure 2A . ( B ) Crystal structure of the NCP containing 601 sequence (PDB: 3LZ0). The positions of the 1-nt gaps are shown as red spheres. ( C ) Bar graphs representing the fraction of processive Pol β molecules in DNA and NCP reactions at 60 mM and 150 mM ionic strength. The F p values at 60 mM ionic strength are 0.46 ± 0.05 and 0.1 ± 0.03 for DNA and NCP, respectively. The F p values at 150 mM ionic strength are 0.12 ± 0.03 and 0.08 ± 0.05 for DNA and NCP, respectively. The error bars report the mean and standard deviation from at least two measurements performed with two independent NCP preparations. ( D ) Bar graph representing the k cat (for total product formation) for DNA and NCP at 60 and 150 mM ionic strength. The k cat values at 60 mM ionic strength are 0.38 ± 0.07 s −1 and 0.02 ± 0.002 s −1 for DNA and NCP, respectively. The k cat values at 150 mM ionic strength are 0.41 ± 0.09 s −1 and 0.02 ± 0.002 s −1 for DNA and NCP, respectively. The error bars report the mean and standard deviation from at least two measurements performed with two independent NCP preparations. ( E ) Single-turnover time-courses for Pol β catalyzed nucleotide insertion at site two. Reaction conditions include 2 μM Pol β with 50 nM DNA (closed) and NCP (open), 150 mM ionic strength, 50 μM dGTP, and standard reaction conditions. The non-zero y-intercept for the NCP time-course is proposed to be a result of the NCP preparation containing a fraction of free DNA or a population of NCP that reacts before the major phase. The data are fit to a single-exponential equation yielding k obs values of 3.0 ± 0.2 s −1 and 0.006 ± 0.001 s −1 for DNA and NCP, respectively.
    Figure Legend Snippet: Pol β catalyzed nucleotide insertion is reduced in the 601 NCP resulting in a lack of processivity. ( A ) NCP processive substrate design. Schematic depicting positions of 1-nt gaps and labeling strategy. Oligonucleotide B is 5΄ labeled with a 32 P phosphate. Site one is located 15 nts and site two 32 nts from the 5΄ end. Reactions were performed as described in Figure 2A . ( B ) Crystal structure of the NCP containing 601 sequence (PDB: 3LZ0). The positions of the 1-nt gaps are shown as red spheres. ( C ) Bar graphs representing the fraction of processive Pol β molecules in DNA and NCP reactions at 60 mM and 150 mM ionic strength. The F p values at 60 mM ionic strength are 0.46 ± 0.05 and 0.1 ± 0.03 for DNA and NCP, respectively. The F p values at 150 mM ionic strength are 0.12 ± 0.03 and 0.08 ± 0.05 for DNA and NCP, respectively. The error bars report the mean and standard deviation from at least two measurements performed with two independent NCP preparations. ( D ) Bar graph representing the k cat (for total product formation) for DNA and NCP at 60 and 150 mM ionic strength. The k cat values at 60 mM ionic strength are 0.38 ± 0.07 s −1 and 0.02 ± 0.002 s −1 for DNA and NCP, respectively. The k cat values at 150 mM ionic strength are 0.41 ± 0.09 s −1 and 0.02 ± 0.002 s −1 for DNA and NCP, respectively. The error bars report the mean and standard deviation from at least two measurements performed with two independent NCP preparations. ( E ) Single-turnover time-courses for Pol β catalyzed nucleotide insertion at site two. Reaction conditions include 2 μM Pol β with 50 nM DNA (closed) and NCP (open), 150 mM ionic strength, 50 μM dGTP, and standard reaction conditions. The non-zero y-intercept for the NCP time-course is proposed to be a result of the NCP preparation containing a fraction of free DNA or a population of NCP that reacts before the major phase. The data are fit to a single-exponential equation yielding k obs values of 3.0 ± 0.2 s −1 and 0.006 ± 0.001 s −1 for DNA and NCP, respectively.

    Techniques Used: Labeling, Sequencing, Standard Deviation

    Models of Pol β searching and gap recognition. ( A ) Proposed modes of Pol β substrate search and recognition. The dark blue circle represents DNA and is positioned such that the viewpoint is looking down DNA. Pol β is shown as a red cartoon. In the searching mode, Pol β is proposed to mainly use its lyase domain to scan DNA in a hopping mechanism. Once a gap is encountered, the lyase domain makes specific interactions with gapped DNA, allowing for the 31-kDa domain to engage. ( B ) The probability of Pol β locating and catalyzing nucleotide insertion within a single DNA binding encounter near predicted physiological ionic strength. The curve represents the fit to the hopping equation shown in Figure 5 . The y-axis represents the probability, or fraction processive (0-1), of Pol β successfully locating and catalyzing nucleotide insertion. The bottom x-axis represents the number of base pairs and the top axis is the corresponding distance (nm). An approximant DNA linker length of 56 bps is shown for reference. This model can be most easily interpreted by using the origin as the site of DNA damage (a 1-nt gap). A horizontal line ∼6 bp down and up-stream from the origin at a probability of 0.75 indicates that when Pol β binds within 6 bp of damage it has a 0.75 chance of catalyzing nucleotide insertion ( Supplementary Figure S4 ). If Pol β binds ∼10 bp down or up-stream from a 1-nt gap it has ∼50% chance of locating and catalyzing nucleotide insertion. Of the Pol β molecules that are processive, 50% will travel a distance of ∼12 bp down or up-stream, resulting in a mean search footprint of ∼24 bp.
    Figure Legend Snippet: Models of Pol β searching and gap recognition. ( A ) Proposed modes of Pol β substrate search and recognition. The dark blue circle represents DNA and is positioned such that the viewpoint is looking down DNA. Pol β is shown as a red cartoon. In the searching mode, Pol β is proposed to mainly use its lyase domain to scan DNA in a hopping mechanism. Once a gap is encountered, the lyase domain makes specific interactions with gapped DNA, allowing for the 31-kDa domain to engage. ( B ) The probability of Pol β locating and catalyzing nucleotide insertion within a single DNA binding encounter near predicted physiological ionic strength. The curve represents the fit to the hopping equation shown in Figure 5 . The y-axis represents the probability, or fraction processive (0-1), of Pol β successfully locating and catalyzing nucleotide insertion. The bottom x-axis represents the number of base pairs and the top axis is the corresponding distance (nm). An approximant DNA linker length of 56 bps is shown for reference. This model can be most easily interpreted by using the origin as the site of DNA damage (a 1-nt gap). A horizontal line ∼6 bp down and up-stream from the origin at a probability of 0.75 indicates that when Pol β binds within 6 bp of damage it has a 0.75 chance of catalyzing nucleotide insertion ( Supplementary Figure S4 ). If Pol β binds ∼10 bp down or up-stream from a 1-nt gap it has ∼50% chance of locating and catalyzing nucleotide insertion. Of the Pol β molecules that are processive, 50% will travel a distance of ∼12 bp down or up-stream, resulting in a mean search footprint of ∼24 bp.

    Techniques Used: Binding Assay

    Pol β uses the positively charged 8-kDa lyase domain for processive searching. Crystal structure of Pol β bound to a 1-nt gapped DNA (PDB 3ISB). ( A ) The lysine residues mutated to alanine are shown as red sticks. Lysines 35, 68 and 72 reside in the lyase domain (yellow) and K113 within the 31-kDa domain (gray). ( B ) The fraction processive at 100 mM ionic strength was measured using the P20 substrate with each mutant Pol β under standard reaction conditions. The mean and standard deviation is shown for three independent experiments. ( C ) Single-turnover analysis for Pol β catalyzed nucleotide insertion measured with indicated mutant enzymes at 100 mM ionic strength. The Wt data is shown as black circles, KΔ3A as red squares, KΔ3R as blue circles, and K113A as green open circles. The nucleotide insertion rate constant ( k pol ) is comparable among the variant enzymes: Wt (2.8 ± 0.2 s −1 ), KΔ3A (2.3 ± 0.2 s −1 ), KΔ3R (2.8 ± 0.2 s −1 ) and K113A (4.4 ± 0.2 s −1 ).
    Figure Legend Snippet: Pol β uses the positively charged 8-kDa lyase domain for processive searching. Crystal structure of Pol β bound to a 1-nt gapped DNA (PDB 3ISB). ( A ) The lysine residues mutated to alanine are shown as red sticks. Lysines 35, 68 and 72 reside in the lyase domain (yellow) and K113 within the 31-kDa domain (gray). ( B ) The fraction processive at 100 mM ionic strength was measured using the P20 substrate with each mutant Pol β under standard reaction conditions. The mean and standard deviation is shown for three independent experiments. ( C ) Single-turnover analysis for Pol β catalyzed nucleotide insertion measured with indicated mutant enzymes at 100 mM ionic strength. The Wt data is shown as black circles, KΔ3A as red squares, KΔ3R as blue circles, and K113A as green open circles. The nucleotide insertion rate constant ( k pol ) is comparable among the variant enzymes: Wt (2.8 ± 0.2 s −1 ), KΔ3A (2.3 ± 0.2 s −1 ), KΔ3R (2.8 ± 0.2 s −1 ) and K113A (4.4 ± 0.2 s −1 ).

    Techniques Used: Mutagenesis, Standard Deviation, Variant Assay

    11) Product Images from "Single molecule counting and assessment of random molecular tagging errors with transposable giga-scale error-correcting barcodes"

    Article Title: Single molecule counting and assessment of random molecular tagging errors with transposable giga-scale error-correcting barcodes

    Journal: BMC Genomics

    doi: 10.1186/s12864-017-4141-4

    Overview of EXB-based molecular barcoding. a Structure of the EXB adapter. The adapter consists of a paired-end Y-adapter structure followed by a 6 bp random nucleotide sequence and three rationally designed 6 bp barcode subunits separated by distinct scaffold sequences. The 6 bp barcode subunits are random combinations of 64 possible sequences as output from the linear generator matrix as shown. The Tn5 transposase recognition sequence at the end of the adapter allows for the generation of sequencing libraries via in vitro Tn5 transposition. b Edit (substitution) distance metrics for all possible 6 bp barcode pairs. Over 93% of pairwise comparisons between barcodes have an edit distance greater than 4. c Schematic of in vitro transposition of EXBs. Tn5 transposase loaded with EXB adapters are incubated with double stranded cDNA. A gap-fill repair reaction then generates paired-end EXB sequencing libraries. After PCR, EXBs are read as inline barcodes, after which the insert sequence is read. d Single-end abundance of EXBs. Single-ended EXB identities were measured by pooling one million reads of each library
    Figure Legend Snippet: Overview of EXB-based molecular barcoding. a Structure of the EXB adapter. The adapter consists of a paired-end Y-adapter structure followed by a 6 bp random nucleotide sequence and three rationally designed 6 bp barcode subunits separated by distinct scaffold sequences. The 6 bp barcode subunits are random combinations of 64 possible sequences as output from the linear generator matrix as shown. The Tn5 transposase recognition sequence at the end of the adapter allows for the generation of sequencing libraries via in vitro Tn5 transposition. b Edit (substitution) distance metrics for all possible 6 bp barcode pairs. Over 93% of pairwise comparisons between barcodes have an edit distance greater than 4. c Schematic of in vitro transposition of EXBs. Tn5 transposase loaded with EXB adapters are incubated with double stranded cDNA. A gap-fill repair reaction then generates paired-end EXB sequencing libraries. After PCR, EXBs are read as inline barcodes, after which the insert sequence is read. d Single-end abundance of EXBs. Single-ended EXB identities were measured by pooling one million reads of each library

    Techniques Used: Sequencing, In Vitro, Incubation, Polymerase Chain Reaction

    12) Product Images from "SMAR1 inhibits Wnt/β-catenin signaling and prevents colorectal cancer progression"

    Article Title: SMAR1 inhibits Wnt/β-catenin signaling and prevents colorectal cancer progression

    Journal: Oncotarget

    doi: 10.18632/oncotarget.25093

    SMAR1 suppresses β-catenin promoter activities FACS analysis of pEGFP1-β-catenin GFP expression ( n = 3, SD) after; ( A ) Co-transfection with FLAG-vector or FLAG-SMAR1, and ( B ) Treatment with 200 ng/mL rh Wnt3a ligand. ( C ) ChIP showing occupancy of SMAR1, HDAC5 and H3K9 Ac (mean ± SD, n = 3) after Wnt3a CM stimulation. ( D ) ChIP showing occupancy of SMAR1, HDAC5 and H3K9 Ac (mean ± SD, n = 3) after SMAR1 overexpression or knockdown. ( E ) ChIP showing occupancy of HDAC5 (mean ± SD, n = 3) after si-HDAC5 knockdown in HCT116 cells. ( F ) Expression of β-catenin after knockdown with 2μg si-HDAC5 plasmid. ( G ) Immunoprecipitation of SMAR1 with HDAC5. ( H ) Immunoprecipitation of HDAC5 with various truncations of SMAR1. ( I ) Immunoprecipitation of HDAC5 with SMAR1 after Wnt3a CM stimulation.
    Figure Legend Snippet: SMAR1 suppresses β-catenin promoter activities FACS analysis of pEGFP1-β-catenin GFP expression ( n = 3, SD) after; ( A ) Co-transfection with FLAG-vector or FLAG-SMAR1, and ( B ) Treatment with 200 ng/mL rh Wnt3a ligand. ( C ) ChIP showing occupancy of SMAR1, HDAC5 and H3K9 Ac (mean ± SD, n = 3) after Wnt3a CM stimulation. ( D ) ChIP showing occupancy of SMAR1, HDAC5 and H3K9 Ac (mean ± SD, n = 3) after SMAR1 overexpression or knockdown. ( E ) ChIP showing occupancy of HDAC5 (mean ± SD, n = 3) after si-HDAC5 knockdown in HCT116 cells. ( F ) Expression of β-catenin after knockdown with 2μg si-HDAC5 plasmid. ( G ) Immunoprecipitation of SMAR1 with HDAC5. ( H ) Immunoprecipitation of HDAC5 with various truncations of SMAR1. ( I ) Immunoprecipitation of HDAC5 with SMAR1 after Wnt3a CM stimulation.

    Techniques Used: FACS, Expressing, Cotransfection, Plasmid Preparation, Chromatin Immunoprecipitation, Over Expression, Immunoprecipitation

    13) Product Images from "RNA Polymerase II Transcription Attenuation at the Yeast DNA Repair Gene, DEF1, Involves Sen1-Dependent and Polyadenylation Site-Dependent Termination"

    Article Title: RNA Polymerase II Transcription Attenuation at the Yeast DNA Repair Gene, DEF1, Involves Sen1-Dependent and Polyadenylation Site-Dependent Termination

    Journal: G3: Genes|Genomes|Genetics

    doi: 10.1534/g3.118.200072

    DEF1 promoter-proximal pA site (pA 1 ) is sufficient to confer Pol II transcription attenuation in a CUP1 /lacZ reporter assay. (A) Schematic of CUP1/lacZ reporter gene used to measure transcription termination (not to scale). The pGAC24 plasmid contains the actin exon(E1)-intron-exon(E2) fused to a CUP1 or lacZ reporter gene. The promoter-proximal DEF1 pA site, CYC1 pA site, or SNR13 transcription termination site were inserted within the intron. The DEF1 attenuator includes the 5′-UTR and upstream ORF but not the consensus TATA box promoter element. In the absence of a pA/terminator insert (No Term.), full-length mRNA production confers copper-resistance and high β-galactosidase activity. In the presence of a pA/terminator insert, attenuated non-coding RNA (ncRNA) production confers copper-sensitivity and low β-galactosidase activity. Trans -acting mutants that prevent pA/terminator recognition promote copper-resistance and higher β-galactosidase activity. (B) DEF1 pA 1 site confers copper-sensitivity in a DEF1-CUP1 reporter, and sen1 , ssu72 , and hrp1 mutants confer copper-resistance. (C) DEF1 pA 1 site reduces expression of CUP1 mRNA due to accumulation of attenuated ncRNA. Note that based on the RT-PCR primer locations (F1, R1, and R2 in panel A), the RT-PCR product from spliced mRNA (231 bp) is shorter than the PCR product from attenuated, unspliced transcript (387 bp). The % attenuated vs. full-length was determined by adding up signal intensities for both bands and determining the relative ratio. No Term. = No Terminator. (D) DEF1 pA 1 site reduces expression of a lacZ reporter similarly to known transcription terminators from CYC1 and SNR13 . β-galactosidase activity was measured following cell lysis and incubation with ONPG substrate, using absorption at OD 600 for cell density and OD 420 for product production. Experiments were performed in biological triplicate, and errors bars show standard deviation.
    Figure Legend Snippet: DEF1 promoter-proximal pA site (pA 1 ) is sufficient to confer Pol II transcription attenuation in a CUP1 /lacZ reporter assay. (A) Schematic of CUP1/lacZ reporter gene used to measure transcription termination (not to scale). The pGAC24 plasmid contains the actin exon(E1)-intron-exon(E2) fused to a CUP1 or lacZ reporter gene. The promoter-proximal DEF1 pA site, CYC1 pA site, or SNR13 transcription termination site were inserted within the intron. The DEF1 attenuator includes the 5′-UTR and upstream ORF but not the consensus TATA box promoter element. In the absence of a pA/terminator insert (No Term.), full-length mRNA production confers copper-resistance and high β-galactosidase activity. In the presence of a pA/terminator insert, attenuated non-coding RNA (ncRNA) production confers copper-sensitivity and low β-galactosidase activity. Trans -acting mutants that prevent pA/terminator recognition promote copper-resistance and higher β-galactosidase activity. (B) DEF1 pA 1 site confers copper-sensitivity in a DEF1-CUP1 reporter, and sen1 , ssu72 , and hrp1 mutants confer copper-resistance. (C) DEF1 pA 1 site reduces expression of CUP1 mRNA due to accumulation of attenuated ncRNA. Note that based on the RT-PCR primer locations (F1, R1, and R2 in panel A), the RT-PCR product from spliced mRNA (231 bp) is shorter than the PCR product from attenuated, unspliced transcript (387 bp). The % attenuated vs. full-length was determined by adding up signal intensities for both bands and determining the relative ratio. No Term. = No Terminator. (D) DEF1 pA 1 site reduces expression of a lacZ reporter similarly to known transcription terminators from CYC1 and SNR13 . β-galactosidase activity was measured following cell lysis and incubation with ONPG substrate, using absorption at OD 600 for cell density and OD 420 for product production. Experiments were performed in biological triplicate, and errors bars show standard deviation.

    Techniques Used: Reporter Assay, Plasmid Preparation, Activity Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Lysis, Incubation, Standard Deviation

    14) Product Images from "Intracellular Survival of Neisseria gonorrhoeae in Male Urethral Epithelial Cells: Importance of a Hexaacyl Lipid A"

    Article Title: Intracellular Survival of Neisseria gonorrhoeae in Male Urethral Epithelial Cells: Importance of a Hexaacyl Lipid A

    Journal: Infection and Immunity

    doi:

    A deletion insertion mutant was made in the N. meningitidis msbB gene. (A) The N. meningitidis PCR product from the pNMBA11 plasmid was cloned into Xba I- Hin dIII-restricted pUC19. (B) A deletion was made in the msbB gene by restriction with Bcl I and Bss HII. (C) The pNMBA11K3 plasmid was generated by ligating the kanamycin resistance gene, aphA -3, into the sites of deletion of the msbB gene.
    Figure Legend Snippet: A deletion insertion mutant was made in the N. meningitidis msbB gene. (A) The N. meningitidis PCR product from the pNMBA11 plasmid was cloned into Xba I- Hin dIII-restricted pUC19. (B) A deletion was made in the msbB gene by restriction with Bcl I and Bss HII. (C) The pNMBA11K3 plasmid was generated by ligating the kanamycin resistance gene, aphA -3, into the sites of deletion of the msbB gene.

    Techniques Used: Mutagenesis, Polymerase Chain Reaction, Plasmid Preparation, Clone Assay, Generated

    15) Product Images from "Rapid modification of the pET-28 expression vector for ligation independent cloning using homologous recombination in Saccharomyces cerevisiae"

    Article Title: Rapid modification of the pET-28 expression vector for ligation independent cloning using homologous recombination in Saccharomyces cerevisiae

    Journal: Plasmid

    doi: 10.1016/j.plasmid.2014.09.005

    Construction of pGAY-28. The modification of pET-28 to replace the multiple cloning region (MCR) with a LIC cassette was accomplished in five steps. In step (1), the parent pET-28 vector is amplified in three segments: A, B, and C. Segment A contains a region homologous to the 3′-end of the linearized yeast shuttle vector YEpADH2p (Y-3′). Segment B contains the LIC cassette at its 3′-end. Segment C contains the LIC cassette at its 5′-end, and a region homologous to the 5′-end of YEpADH2p (Y-5′). In step (2), transformation of linearized YEpADH2p and the three amplified segments into competent S. cerevisiae leads to step (3), where the overlapping segments undergo homologous recombination in vivo . In step (4), two of the original primers from step (1) are used again to amplify the modified expression vector using “colony PCR”. Since these primers were originally designed to anneal upstream of a single XmaI restriction site, step (5) involves digestion of the amplicon with XmaI followed by treatment with DNA ligase, yielding the complete pGAY-28 expression vector.
    Figure Legend Snippet: Construction of pGAY-28. The modification of pET-28 to replace the multiple cloning region (MCR) with a LIC cassette was accomplished in five steps. In step (1), the parent pET-28 vector is amplified in three segments: A, B, and C. Segment A contains a region homologous to the 3′-end of the linearized yeast shuttle vector YEpADH2p (Y-3′). Segment B contains the LIC cassette at its 3′-end. Segment C contains the LIC cassette at its 5′-end, and a region homologous to the 5′-end of YEpADH2p (Y-5′). In step (2), transformation of linearized YEpADH2p and the three amplified segments into competent S. cerevisiae leads to step (3), where the overlapping segments undergo homologous recombination in vivo . In step (4), two of the original primers from step (1) are used again to amplify the modified expression vector using “colony PCR”. Since these primers were originally designed to anneal upstream of a single XmaI restriction site, step (5) involves digestion of the amplicon with XmaI followed by treatment with DNA ligase, yielding the complete pGAY-28 expression vector.

    Techniques Used: Modification, Positron Emission Tomography, Clone Assay, Plasmid Preparation, Amplification, Transformation Assay, Homologous Recombination, In Vivo, Expressing, Polymerase Chain Reaction

    16) Product Images from "Development and Evaluation of an Enzyme-Linked Immunosorbent Assay Based on Recombinant VP2 Capsids for the Detection of Antibodies to Aleutian Mink Disease Virus "

    Article Title: Development and Evaluation of an Enzyme-Linked Immunosorbent Assay Based on Recombinant VP2 Capsids for the Detection of Antibodies to Aleutian Mink Disease Virus

    Journal: Clinical and Vaccine Immunology : CVI

    doi: 10.1128/CVI.00148-09

    Negative-stain electron micrograph of recombinant AMDV VP2 VLPs. The white bar at the bottom indicates 200 nm.
    Figure Legend Snippet: Negative-stain electron micrograph of recombinant AMDV VP2 VLPs. The white bar at the bottom indicates 200 nm.

    Techniques Used: Staining, Recombinant

    SDS-PAGE gel (A) and Western blot (B) of recombinant AMDV VP2 protein with sera from ADMV antibody-positive mink. Lanes: 1 and 5, molecular-mass marker; 2, S. frugiperda 9 cells; 3, recombinant baculovirus-infected Sf9 cells; 4, purified recombinant antigen;
    Figure Legend Snippet: SDS-PAGE gel (A) and Western blot (B) of recombinant AMDV VP2 protein with sera from ADMV antibody-positive mink. Lanes: 1 and 5, molecular-mass marker; 2, S. frugiperda 9 cells; 3, recombinant baculovirus-infected Sf9 cells; 4, purified recombinant antigen;

    Techniques Used: SDS Page, Western Blot, Recombinant, Marker, Infection, Purification

    17) Product Images from "The Origin of 8-Amino-3,8-dideoxy-d-manno-octulosonic Acid (Kdo8N) in the Lipopolysaccharide of Shewanella oneidensis *"

    Article Title: The Origin of 8-Amino-3,8-dideoxy-d-manno-octulosonic Acid (Kdo8N) in the Lipopolysaccharide of Shewanella oneidensis *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M113.453324

    TLC analysis of lipid A from E. coli expressing potential Kdo8N biosynthetic genes. Lane A , WBB06 expressing empty pWSK29 control; lane B , WBB06 expressing kdnA , kdnB , and kdsB . Lipid A samples were extracted, run on silica TLC plates in 25:15:3.5:4 CHCl
    Figure Legend Snippet: TLC analysis of lipid A from E. coli expressing potential Kdo8N biosynthetic genes. Lane A , WBB06 expressing empty pWSK29 control; lane B , WBB06 expressing kdnA , kdnB , and kdsB . Lipid A samples were extracted, run on silica TLC plates in 25:15:3.5:4 CHCl

    Techniques Used: Thin Layer Chromatography, Expressing

    18) Product Images from "Isolation and Characterization of a Rolling-Circle-Type Plasmid from Rhodococcus erythropolis and Application of the Plasmid to Multiple-Recombinant-Protein Expression"

    Article Title: Isolation and Characterization of a Rolling-Circle-Type Plasmid from Rhodococcus erythropolis and Application of the Plasmid to Multiple-Recombinant-Protein Expression

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.70.9.5557-5568.2004

    Coexpression and purification of six-His-GFP and six-His-PIP. R. erythropolis JCM3201 was cotransformed with pHN425 and pHN389 (lanes 1 and 2) and with pHN426 and pHN409 (lanes 3 and 4). As controls, R. erythropolis JCM3201 was also transformed with either pHN425 (lanes 5 and 6), pHN426 (lanes 7 and 8), pHN389 (lanes 9 and 10), or pHN409 (lanes 11 and 12). Expressed GFP and/or PIP were purified by Ni-nitrilotriacetic acid superflow (see Materials and Methods). Each sample was prepared from 50 ml of culture medium. Cell extracts (odd-numbered lanes) and purified proteins (even-numbered lanes) were analyzed by sodium dodecyl sulfate-12% polyacrylamide gel electrophoresis followed by staining of the gel with Coomassie brilliant blue G-250.
    Figure Legend Snippet: Coexpression and purification of six-His-GFP and six-His-PIP. R. erythropolis JCM3201 was cotransformed with pHN425 and pHN389 (lanes 1 and 2) and with pHN426 and pHN409 (lanes 3 and 4). As controls, R. erythropolis JCM3201 was also transformed with either pHN425 (lanes 5 and 6), pHN426 (lanes 7 and 8), pHN389 (lanes 9 and 10), or pHN409 (lanes 11 and 12). Expressed GFP and/or PIP were purified by Ni-nitrilotriacetic acid superflow (see Materials and Methods). Each sample was prepared from 50 ml of culture medium. Cell extracts (odd-numbered lanes) and purified proteins (even-numbered lanes) were analyzed by sodium dodecyl sulfate-12% polyacrylamide gel electrophoresis followed by staining of the gel with Coomassie brilliant blue G-250.

    Techniques Used: Purification, Transformation Assay, Polyacrylamide Gel Electrophoresis, Staining

    19) Product Images from "Genetic Analysis of G Protein-Coupled Receptor Expression in Escherichia coli"

    Article Title: Genetic Analysis of G Protein-Coupled Receptor Expression in Escherichia coli

    Journal: Biotechnology and bioengineering

    doi: 10.1002/bit.22097

    A : Design of the expression vectors pASKCB1 and pASKCB1-GFP. P tet : tetracycline promoter; RBS: ribosome-binding site; FLAG: FLAG octapeptide epitope; CB1: E. coli codon-optimized gene encoding full-length human central cannabinoid receptor; His 8 : octahistidine tag; TEV: recognition sequence for the tobacco etch virus protease; GFP: green fluorescent protein variant GFPmut2 optimized via FACS. B : Schematic representation of the screening process. E. coli MC4100A cells were subjected to transposon mutagneesis, and FACS was employed to screen the generated MC4100A::Tn 5 (pASKCB1-GFP) library and isolate E. coli transposon insertion mutations that confer higher fluorescence due to an increase in the amount of membrane-bound CB1-GFP. For FACS screening, cells were initially gated based on size (gate P1) on a side-scatter (SSC-H) versus forward-scatter (FSC-H) plot. Subsequently, the clones corresponding to the top 1-3% fluorescent events (gate P2) were isolated and subjected to repeated rounds of FACS sorting.
    Figure Legend Snippet: A : Design of the expression vectors pASKCB1 and pASKCB1-GFP. P tet : tetracycline promoter; RBS: ribosome-binding site; FLAG: FLAG octapeptide epitope; CB1: E. coli codon-optimized gene encoding full-length human central cannabinoid receptor; His 8 : octahistidine tag; TEV: recognition sequence for the tobacco etch virus protease; GFP: green fluorescent protein variant GFPmut2 optimized via FACS. B : Schematic representation of the screening process. E. coli MC4100A cells were subjected to transposon mutagneesis, and FACS was employed to screen the generated MC4100A::Tn 5 (pASKCB1-GFP) library and isolate E. coli transposon insertion mutations that confer higher fluorescence due to an increase in the amount of membrane-bound CB1-GFP. For FACS screening, cells were initially gated based on size (gate P1) on a side-scatter (SSC-H) versus forward-scatter (FSC-H) plot. Subsequently, the clones corresponding to the top 1-3% fluorescent events (gate P2) were isolated and subjected to repeated rounds of FACS sorting.

    Techniques Used: Expressing, Binding Assay, FLAG-tag, Sequencing, Variant Assay, FACS, Generated, Fluorescence, Clone Assay, Isolation

    Identification of transposon insertions that alleviate cell toxicity associated with CB1 production that do not map on dnaJ . A : Fluorescence histograms of MC4100A, GS102 (MC4100A dinG ::Tn 5 ), and GS103 ((MC4100A nhaR ::Tn 5 ) cells expressing CB1-GFP at room temperature for approximately 5 h. Histograms correspond to a total population of 10,000 cells. M: arithmetic mean; a.u: arbitrary units. B : Cell density of MC4100A, GS102 and GS103 cultures expressing CB1-GFP at room temperature for approximately 5 h. The reported values correspond to the average of four replica experiments and the error bars represent one standard deviation from the mean value. OD 600 : optical density at 600 nm. C : Western blots on total cell lysates demonstrating the production levels of DnaJ in MC4100A, GS101, GS102, and GS103 cells. Cells were grown to mid-log phase at 37°C and DnaJ production levels were probed with an anti-DnaJ antibody. An equal number of cells were loaded on each lane as judged by OD 600 ]
    Figure Legend Snippet: Identification of transposon insertions that alleviate cell toxicity associated with CB1 production that do not map on dnaJ . A : Fluorescence histograms of MC4100A, GS102 (MC4100A dinG ::Tn 5 ), and GS103 ((MC4100A nhaR ::Tn 5 ) cells expressing CB1-GFP at room temperature for approximately 5 h. Histograms correspond to a total population of 10,000 cells. M: arithmetic mean; a.u: arbitrary units. B : Cell density of MC4100A, GS102 and GS103 cultures expressing CB1-GFP at room temperature for approximately 5 h. The reported values correspond to the average of four replica experiments and the error bars represent one standard deviation from the mean value. OD 600 : optical density at 600 nm. C : Western blots on total cell lysates demonstrating the production levels of DnaJ in MC4100A, GS101, GS102, and GS103 cells. Cells were grown to mid-log phase at 37°C and DnaJ production levels were probed with an anti-DnaJ antibody. An equal number of cells were loaded on each lane as judged by OD 600 ]

    Techniques Used: Fluorescence, Expressing, Standard Deviation, Western Blot

    Transposon mutagenesis and FACS screening on the GS104 (MC4100A Δ dinG ) strain background. A : Enrichment of the MC4100A Δ dinG ::Tn 5 (pASKCB1-GFP) library with higher-fluorescence clones after repeated rounds of FACS sorting and the identification of the strain GS105 (MC4100A Δ dinG dnaJ ::Tn 5 ) exhibiting markedly higher CB1-GFP fluorescence. Fluorescence histograms correspond to a total population of 10,000 cells. M: arithmetic mean; a.u: arbitrary units. B : Comparison of the cell density at saturation of parental MC4100A and GS105 cells expressing CB1-GFP. The reported data correspond to the average of four replica experiments and the error bars represent one standard deviation from the mean value. OD 600 : optical density at 600 nm. C : Comparison of the production of membrane-integrated CB1-GFP fusion in parental MC4100A, GS101, and GS105 cells normalized by number of cells ( left ) and by unit volume of bacterial culture ( right ) with Western blotting. D ]
    Figure Legend Snippet: Transposon mutagenesis and FACS screening on the GS104 (MC4100A Δ dinG ) strain background. A : Enrichment of the MC4100A Δ dinG ::Tn 5 (pASKCB1-GFP) library with higher-fluorescence clones after repeated rounds of FACS sorting and the identification of the strain GS105 (MC4100A Δ dinG dnaJ ::Tn 5 ) exhibiting markedly higher CB1-GFP fluorescence. Fluorescence histograms correspond to a total population of 10,000 cells. M: arithmetic mean; a.u: arbitrary units. B : Comparison of the cell density at saturation of parental MC4100A and GS105 cells expressing CB1-GFP. The reported data correspond to the average of four replica experiments and the error bars represent one standard deviation from the mean value. OD 600 : optical density at 600 nm. C : Comparison of the production of membrane-integrated CB1-GFP fusion in parental MC4100A, GS101, and GS105 cells normalized by number of cells ( left ) and by unit volume of bacterial culture ( right ) with Western blotting. D ]

    Techniques Used: Mutagenesis, FACS, Fluorescence, Clone Assay, Expressing, Standard Deviation, Western Blot

    20) Product Images from "Secretion of functional formate dehydrogenase in Pichia pastoris"

    Article Title: Secretion of functional formate dehydrogenase in Pichia pastoris

    Journal: Protein Engineering, Design and Selection

    doi: 10.1093/protein/gzx010

    SDS-PAGE (10% acrylamide) analysis of media after growing yeast with empty pPIC9 for 120 h (Lane 1); media after growing yeast with FDH-C for 0 h (Lane 2), 8 h (Lane 3), 24 h (Lane 4), 48 h (Lane 5), 72 h (Lane 6), 96 h (Lane 7) and 120 h (Lane 8).
    Figure Legend Snippet: SDS-PAGE (10% acrylamide) analysis of media after growing yeast with empty pPIC9 for 120 h (Lane 1); media after growing yeast with FDH-C for 0 h (Lane 2), 8 h (Lane 3), 24 h (Lane 4), 48 h (Lane 5), 72 h (Lane 6), 96 h (Lane 7) and 120 h (Lane 8).

    Techniques Used: SDS Page

    21) Product Images from "Inactivation of the Major Hemolysin Gene Influences Expression of the Nonribosomal Peptide Synthetase Gene swrA in the Insect Pathogen Serratia sp. Strain SCBI"

    Article Title: Inactivation of the Major Hemolysin Gene Influences Expression of the Nonribosomal Peptide Synthetase Gene swrA in the Insect Pathogen Serratia sp. Strain SCBI

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.00333-17

    Loss of shlA results in a significant reduction or loss of hemolytic activity in Serratia sp. SCBI. (A) Hemolytic activity against SRBCs by wild-type Serratia sp. SCBI and five hemolysin mutants (≥4.0 × 10 6 CFU) was measured over 4 h at 28°C. Error bars represent standard deviations of the results from at least two independent experiments. (B) Representation of the results of rescue cloning the hemolysis mutants; 5 of the 7 hemolysis mutants had the transposon insertion in shlA , and all hit in a different location within the gene.
    Figure Legend Snippet: Loss of shlA results in a significant reduction or loss of hemolytic activity in Serratia sp. SCBI. (A) Hemolytic activity against SRBCs by wild-type Serratia sp. SCBI and five hemolysin mutants (≥4.0 × 10 6 CFU) was measured over 4 h at 28°C. Error bars represent standard deviations of the results from at least two independent experiments. (B) Representation of the results of rescue cloning the hemolysis mutants; 5 of the 7 hemolysis mutants had the transposon insertion in shlA , and all hit in a different location within the gene.

    Techniques Used: Activity Assay, Clone Assay

    Mutants with transposon insertions in shlA had significantly increased mRNA levels of the 17,775-bp NRPS swrA when swarming. qRT-PCR was performed on RNA extracted from swarmer cells on 0.65% agar PP3 plates. mRNA levels were normalized to the level of the l21p housekeeping gene and compared to the calibrator, wild-type Serratia sp. SCBI (WT SCBI). Data are presented as the relative changes in gene expression from those of the calibrator obtained under the test conditions. (A) Comparison of flhD , fliC , and swrA mRNA levels when swarm ring diameters were at 40 mm between wild-type Serratia sp. SCBI and the five shlA mutants; (B) mRNA levels of flhD , fliC , and swrA by wild-type Serratia sp. SCBI and shlA mutant 6-E3 at swarm ring diameters of 20, 40, and 80 mm. Error bars represent standard deviations of the results from at least two independent experiments. *, P
    Figure Legend Snippet: Mutants with transposon insertions in shlA had significantly increased mRNA levels of the 17,775-bp NRPS swrA when swarming. qRT-PCR was performed on RNA extracted from swarmer cells on 0.65% agar PP3 plates. mRNA levels were normalized to the level of the l21p housekeeping gene and compared to the calibrator, wild-type Serratia sp. SCBI (WT SCBI). Data are presented as the relative changes in gene expression from those of the calibrator obtained under the test conditions. (A) Comparison of flhD , fliC , and swrA mRNA levels when swarm ring diameters were at 40 mm between wild-type Serratia sp. SCBI and the five shlA mutants; (B) mRNA levels of flhD , fliC , and swrA by wild-type Serratia sp. SCBI and shlA mutant 6-E3 at swarm ring diameters of 20, 40, and 80 mm. Error bars represent standard deviations of the results from at least two independent experiments. *, P

    Techniques Used: Quantitative RT-PCR, Expressing, Mutagenesis

    Mutations in swrA had various effects on antibiotic activity, and these effects were dependent on the site of transposon insertion in swrA . (A) Rescue cloning showed that mutants 1-A4, 13-G2, and 11-B8 had the transposon insertion at different sites within swrA . (B) Domain structure of SwrA. (C) Wild-type Serratia sp. SCBI and the swrA mutants were spot inoculated onto PP3 plates, incubated for 48 h at 28°C, killed by chloroform, and overlaid with warm 0.8% agar containing Micrococcus luteus . Clearing zones were observed following 24 h at 28°C. Results are shown as the averages ± standard deviations of 9 measurements from 3 independent experiments.
    Figure Legend Snippet: Mutations in swrA had various effects on antibiotic activity, and these effects were dependent on the site of transposon insertion in swrA . (A) Rescue cloning showed that mutants 1-A4, 13-G2, and 11-B8 had the transposon insertion at different sites within swrA . (B) Domain structure of SwrA. (C) Wild-type Serratia sp. SCBI and the swrA mutants were spot inoculated onto PP3 plates, incubated for 48 h at 28°C, killed by chloroform, and overlaid with warm 0.8% agar containing Micrococcus luteus . Clearing zones were observed following 24 h at 28°C. Results are shown as the averages ± standard deviations of 9 measurements from 3 independent experiments.

    Techniques Used: Activity Assay, Clone Assay, Incubation

    22) Product Images from "Periplasmic protein EipA determines envelope stress resistance and virulence in Brucella abortus"

    Article Title: Periplasmic protein EipA determines envelope stress resistance and virulence in Brucella abortus

    Journal: Molecular microbiology

    doi: 10.1111/mmi.14178

    The essential cell cycle regulator, CtrA, directly binds the promoter region of eipA in B. abortus and activates its expression. A) Electrophoretic mobility shift assay (EMSA) with purified CtrA protein and eipA promoter region (P eipA ). Top: cartoon representation of the eipA chromosomal locus, with ctrA ( bab1_1614 ; white), chpT ( bab1_1613 ; white), and the CtrA binding site (CBS, black rectangle) present in eipA (brown) promoter region. Increasing concentrations of CtrA (9 – 500 nM) were mixed with 0.1 ng of radiolabelled DNA corresponding to eipA promoter region (131 bp) (lane 1 to 7). A full shift of the DNA was observed at 500 nM CtrA. Lane 8 shows the DNA alone, without CtrA (0 nM). To test CtrA binding specificity, we competed 0.1 ng of radiolabelled wild-type DNA with 1 ng of unlabelled wild-type DNA (lane 9, (a)) or with 1 ng of unlabeled and mutated DNA (lane 10, (b)). This experiment was independently performed four times; a representative gel is presented. B) Specificity of the rabbit anti-EipA polyclonal serum was tested by western blot using cell lysate from wild-type B. abortus (lane 1), the eipA deletion strain (lane 2) and the complemented (lane 3) strains. Non-specific bands (nsb) were used as loading controls. C) EipA protein levels were evaluated in wild-type B. abortus (lane 1) or in a strain carrying an inducible cpdR D52A overexpression (OE) plasmid without (lane 2) or with (lane 3) IPTG inducer. Western blots of lysates from these strains were probed with anti-EipA serum (top), anti-CtrA serum (middle), and anti-PhyR serum as a loading control (bottom). This experiment was independently repeated three times; a representative gel is presented.
    Figure Legend Snippet: The essential cell cycle regulator, CtrA, directly binds the promoter region of eipA in B. abortus and activates its expression. A) Electrophoretic mobility shift assay (EMSA) with purified CtrA protein and eipA promoter region (P eipA ). Top: cartoon representation of the eipA chromosomal locus, with ctrA ( bab1_1614 ; white), chpT ( bab1_1613 ; white), and the CtrA binding site (CBS, black rectangle) present in eipA (brown) promoter region. Increasing concentrations of CtrA (9 – 500 nM) were mixed with 0.1 ng of radiolabelled DNA corresponding to eipA promoter region (131 bp) (lane 1 to 7). A full shift of the DNA was observed at 500 nM CtrA. Lane 8 shows the DNA alone, without CtrA (0 nM). To test CtrA binding specificity, we competed 0.1 ng of radiolabelled wild-type DNA with 1 ng of unlabelled wild-type DNA (lane 9, (a)) or with 1 ng of unlabeled and mutated DNA (lane 10, (b)). This experiment was independently performed four times; a representative gel is presented. B) Specificity of the rabbit anti-EipA polyclonal serum was tested by western blot using cell lysate from wild-type B. abortus (lane 1), the eipA deletion strain (lane 2) and the complemented (lane 3) strains. Non-specific bands (nsb) were used as loading controls. C) EipA protein levels were evaluated in wild-type B. abortus (lane 1) or in a strain carrying an inducible cpdR D52A overexpression (OE) plasmid without (lane 2) or with (lane 3) IPTG inducer. Western blots of lysates from these strains were probed with anti-EipA serum (top), anti-CtrA serum (middle), and anti-PhyR serum as a loading control (bottom). This experiment was independently repeated three times; a representative gel is presented.

    Techniques Used: Expressing, Electrophoretic Mobility Shift Assay, Purification, Binding Assay, Western Blot, Over Expression, Plasmid Preparation

    23) Product Images from "Attenuation of DNA charge transport by compaction into a nucleosome core particle"

    Article Title: Attenuation of DNA charge transport by compaction into a nucleosome core particle

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl030

    Structural characterization of AQ-157TG rNCPs. ( A ) Exonuclease III footprinting of AQ-157TG rNCPs (lane 1) and free AQ-157TG (lane 2). The restriction of ExoIII activity to the ∼10 bp proximal to AQ in the AQ-157TG rNCPs is evident. ( B ) Autoradiogram of hydroxyl radical footprinting on AQ-157TG rNCPs (lanes 1 and 2) and free AQ-157TG (lane 3). ( C ) Partial scan of the footprint in B of both free AQ-157TG (bottom) and AQ-157TG rNCPs (top). The 10 bp periodic cutting in the rNCPs is apparent.
    Figure Legend Snippet: Structural characterization of AQ-157TG rNCPs. ( A ) Exonuclease III footprinting of AQ-157TG rNCPs (lane 1) and free AQ-157TG (lane 2). The restriction of ExoIII activity to the ∼10 bp proximal to AQ in the AQ-157TG rNCPs is evident. ( B ) Autoradiogram of hydroxyl radical footprinting on AQ-157TG rNCPs (lanes 1 and 2) and free AQ-157TG (lane 3). ( C ) Partial scan of the footprint in B of both free AQ-157TG (bottom) and AQ-157TG rNCPs (top). The 10 bp periodic cutting in the rNCPs is apparent.

    Techniques Used: Footprinting, Activity Assay

    24) Product Images from "7,8-dihydro-8-oxoadenine, a highly mutagenic adduct, is repaired by Escherichia coli and human mismatch-specific uracil/thymine-DNA glycosylases"

    Article Title: 7,8-dihydro-8-oxoadenine, a highly mutagenic adduct, is repaired by Escherichia coli and human mismatch-specific uracil/thymine-DNA glycosylases

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gks1149

    In vitro reconstitution of the BER pathway using 8oxoA•T duplex DNA substrate. 5 nM 40 mer 8oxoA•T duplex was incubated in the presence of 20 nM hTDG, 5 nM APE1, 2 nM FEN1, 0.1 U POL-β and 5 nM T4 DNA ligase in buffer containing 20 µCi of [α- 32 P]dATP, 50 µM dNTPs, 50 mM HEPES–KOH (pH 7.6), 30 mM NaCl, 0.1 mg/ml BSA, 2 mM DTT, 2 mM ATP and 3 mM MgCl 2 for 5 and 30 min at 37°C. Lane 1, 30 min in the absence of hTDG and T4 DNA ligase; lane 2, 5 min in the absence of T4 DNA ligase; lane 3, same as 2, but 30 min; lane 4, 30 min in the presence of all proteins. For details see ‘Materials and Methods’ section.
    Figure Legend Snippet: In vitro reconstitution of the BER pathway using 8oxoA•T duplex DNA substrate. 5 nM 40 mer 8oxoA•T duplex was incubated in the presence of 20 nM hTDG, 5 nM APE1, 2 nM FEN1, 0.1 U POL-β and 5 nM T4 DNA ligase in buffer containing 20 µCi of [α- 32 P]dATP, 50 µM dNTPs, 50 mM HEPES–KOH (pH 7.6), 30 mM NaCl, 0.1 mg/ml BSA, 2 mM DTT, 2 mM ATP and 3 mM MgCl 2 for 5 and 30 min at 37°C. Lane 1, 30 min in the absence of hTDG and T4 DNA ligase; lane 2, 5 min in the absence of T4 DNA ligase; lane 3, same as 2, but 30 min; lane 4, 30 min in the presence of all proteins. For details see ‘Materials and Methods’ section.

    Techniques Used: In Vitro, Incubation

    25) Product Images from "DNA supercoiling, a critical signal regulating the basal expression of the lac operon in Escherichia coli"

    Article Title: DNA supercoiling, a critical signal regulating the basal expression of the lac operon in Escherichia coli

    Journal: Scientific Reports

    doi: 10.1038/srep19243

    One molecule of LacI tetramer divided a supercoiled DNA molecule plasmid pCB126 into two independent topological domains. ( a ) Plasmid pCB126 carrying two lac O1 operators in two different locations was constructed as detailed in Methods. ( b ) The nicking enzyme Nt.BbvCI was able to rapidly digest pCB126. Time course of enzyme digestion of pCB126 using 16 units of Nt.BbvCI in 1 × NEBuffer 4 at 37 °C. Lane 1 contained the undigested scDNA. ( c ) Time course of DNA supercoiling diffusion in the presence of LacI. The DNA-nicking assays were performed as described under Methods. Each reaction mixture (320 μL) contained 0.156 nM of pCB126, 2.5 nM of LacI, and 16 units of Nt.BbvCI. The reactions were incubated at 37 °C for the time indicated. Then a large excess of a double-stranded oligonucleotide contain an Nt.BbvCI recognition site was added to the reaction mixture to inhibit the restriction enzyme activities. The nicked DNA templates were ligated by T4 DNA ligase in the presence of 1 mM of ATP at 37 °C for 5 min and the reactions were terminated by phenol extraction. The DNA molecules were isolated and subjected to agarose gel electrophoresis. ( d ) Quantification analysis of the time course. The percentage of supercoiled DNA was plotted against the reaction time. The curve was generated by fitting the data to a 1st-order rate equation to yield a rate constant of 0.016 sec −1 and a t 1/2 of 52 sec.
    Figure Legend Snippet: One molecule of LacI tetramer divided a supercoiled DNA molecule plasmid pCB126 into two independent topological domains. ( a ) Plasmid pCB126 carrying two lac O1 operators in two different locations was constructed as detailed in Methods. ( b ) The nicking enzyme Nt.BbvCI was able to rapidly digest pCB126. Time course of enzyme digestion of pCB126 using 16 units of Nt.BbvCI in 1 × NEBuffer 4 at 37 °C. Lane 1 contained the undigested scDNA. ( c ) Time course of DNA supercoiling diffusion in the presence of LacI. The DNA-nicking assays were performed as described under Methods. Each reaction mixture (320 μL) contained 0.156 nM of pCB126, 2.5 nM of LacI, and 16 units of Nt.BbvCI. The reactions were incubated at 37 °C for the time indicated. Then a large excess of a double-stranded oligonucleotide contain an Nt.BbvCI recognition site was added to the reaction mixture to inhibit the restriction enzyme activities. The nicked DNA templates were ligated by T4 DNA ligase in the presence of 1 mM of ATP at 37 °C for 5 min and the reactions were terminated by phenol extraction. The DNA molecules were isolated and subjected to agarose gel electrophoresis. ( d ) Quantification analysis of the time course. The percentage of supercoiled DNA was plotted against the reaction time. The curve was generated by fitting the data to a 1st-order rate equation to yield a rate constant of 0.016 sec −1 and a t 1/2 of 52 sec.

    Techniques Used: Plasmid Preparation, Construct, Diffusion-based Assay, Incubation, Isolation, Agarose Gel Electrophoresis, Generated, Size-exclusion Chromatography

    26) Product Images from "Cellular reagents for diagnostics and synthetic biology"

    Article Title: Cellular reagents for diagnostics and synthetic biology

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0201681

    PCR and Gibson assembly using cellular reagents. (a) Schematic depicting cellular PCR followed by cellular Gibson assembly for constructing new plasmids. Bacteria harboring target plasmids are mixed with polymerase-expressing cellular reagents and PCR is initiated by adding appropriate primers, buffer, and dNTP. The resulting PCR products are incubated with cellular reagents expressing Gibson assembly enzymes–Taq DNA polymerase, Taq DNA ligase, and T5 exonuclease–to assemble the new construct. (b) Cellular PCR amplification of vector and insert fragments directly from  E .  coli  bacteria bearing target DNA plasmids using 2 x 10 7  cells of Phusion cellular reagents. Assembly parts include: (i) “pATetO 6XHis full length” vector for two part assembly with Kan r  cassette bearing appropriate overlapping ends, and (ii) “pUC19 Fragments 1 and 2” for three part assembly with Kan r  cassette whose ends overlap with pUC19 vector fragments. (c) Gibson assembly of agarose gel purified and unpurified cellular PCR products using pure or cellular Gibson assembly reagents. In “negative control” samples the PCR products were incubated in Gibson reaction buffer without pure or cellular Gibson enzymes. “pATetO 6XHis + Kan r ”represents a two part Gibson assembly while “Puc19 Fragment 1 + pUC19 Fragment 2 + Kan r ” represents a three-part Gibson assembly. Representative number of clones recovered in each case in the presence of both ampicillin and kanamycin are reported.
    Figure Legend Snippet: PCR and Gibson assembly using cellular reagents. (a) Schematic depicting cellular PCR followed by cellular Gibson assembly for constructing new plasmids. Bacteria harboring target plasmids are mixed with polymerase-expressing cellular reagents and PCR is initiated by adding appropriate primers, buffer, and dNTP. The resulting PCR products are incubated with cellular reagents expressing Gibson assembly enzymes–Taq DNA polymerase, Taq DNA ligase, and T5 exonuclease–to assemble the new construct. (b) Cellular PCR amplification of vector and insert fragments directly from E . coli bacteria bearing target DNA plasmids using 2 x 10 7 cells of Phusion cellular reagents. Assembly parts include: (i) “pATetO 6XHis full length” vector for two part assembly with Kan r cassette bearing appropriate overlapping ends, and (ii) “pUC19 Fragments 1 and 2” for three part assembly with Kan r cassette whose ends overlap with pUC19 vector fragments. (c) Gibson assembly of agarose gel purified and unpurified cellular PCR products using pure or cellular Gibson assembly reagents. In “negative control” samples the PCR products were incubated in Gibson reaction buffer without pure or cellular Gibson enzymes. “pATetO 6XHis + Kan r ”represents a two part Gibson assembly while “Puc19 Fragment 1 + pUC19 Fragment 2 + Kan r ” represents a three-part Gibson assembly. Representative number of clones recovered in each case in the presence of both ampicillin and kanamycin are reported.

    Techniques Used: Polymerase Chain Reaction, Expressing, Incubation, Construct, Amplification, Plasmid Preparation, Agarose Gel Electrophoresis, Purification, Clone Assay

    27) Product Images from "Cellular reagents for diagnostics and synthetic biology"

    Article Title: Cellular reagents for diagnostics and synthetic biology

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0201681

    PCR and Gibson assembly using cellular reagents. (a) Schematic depicting cellular PCR followed by cellular Gibson assembly for constructing new plasmids. Bacteria harboring target plasmids are mixed with polymerase-expressing cellular reagents and PCR is initiated by adding appropriate primers, buffer, and dNTP. The resulting PCR products are incubated with cellular reagents expressing Gibson assembly enzymes–Taq DNA polymerase, Taq DNA ligase, and T5 exonuclease–to assemble the new construct. (b) Cellular PCR amplification of vector and insert fragments directly from  E .  coli  bacteria bearing target DNA plasmids using 2 x 10 7  cells of Phusion cellular reagents. Assembly parts include: (i) “pATetO 6XHis full length” vector for two part assembly with Kan r  cassette bearing appropriate overlapping ends, and (ii) “pUC19 Fragments 1 and 2” for three part assembly with Kan r  cassette whose ends overlap with pUC19 vector fragments. (c) Gibson assembly of agarose gel purified and unpurified cellular PCR products using pure or cellular Gibson assembly reagents. In “negative control” samples the PCR products were incubated in Gibson reaction buffer without pure or cellular Gibson enzymes. “pATetO 6XHis + Kan r ”represents a two part Gibson assembly while “Puc19 Fragment 1 + pUC19 Fragment 2 + Kan r ” represents a three-part Gibson assembly. Representative number of clones recovered in each case in the presence of both ampicillin and kanamycin are reported.
    Figure Legend Snippet: PCR and Gibson assembly using cellular reagents. (a) Schematic depicting cellular PCR followed by cellular Gibson assembly for constructing new plasmids. Bacteria harboring target plasmids are mixed with polymerase-expressing cellular reagents and PCR is initiated by adding appropriate primers, buffer, and dNTP. The resulting PCR products are incubated with cellular reagents expressing Gibson assembly enzymes–Taq DNA polymerase, Taq DNA ligase, and T5 exonuclease–to assemble the new construct. (b) Cellular PCR amplification of vector and insert fragments directly from E . coli bacteria bearing target DNA plasmids using 2 x 10 7 cells of Phusion cellular reagents. Assembly parts include: (i) “pATetO 6XHis full length” vector for two part assembly with Kan r cassette bearing appropriate overlapping ends, and (ii) “pUC19 Fragments 1 and 2” for three part assembly with Kan r cassette whose ends overlap with pUC19 vector fragments. (c) Gibson assembly of agarose gel purified and unpurified cellular PCR products using pure or cellular Gibson assembly reagents. In “negative control” samples the PCR products were incubated in Gibson reaction buffer without pure or cellular Gibson enzymes. “pATetO 6XHis + Kan r ”represents a two part Gibson assembly while “Puc19 Fragment 1 + pUC19 Fragment 2 + Kan r ” represents a three-part Gibson assembly. Representative number of clones recovered in each case in the presence of both ampicillin and kanamycin are reported.

    Techniques Used: Polymerase Chain Reaction, Expressing, Incubation, Construct, Amplification, Plasmid Preparation, Agarose Gel Electrophoresis, Purification, Clone Assay

    28) Product Images from "Cellular reagents for diagnostics and synthetic biology"

    Article Title: Cellular reagents for diagnostics and synthetic biology

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0201681

    PCR and Gibson assembly using cellular reagents. (a) Schematic depicting cellular PCR followed by cellular Gibson assembly for constructing new plasmids. Bacteria harboring target plasmids are mixed with polymerase-expressing cellular reagents and PCR is initiated by adding appropriate primers, buffer, and dNTP. The resulting PCR products are incubated with cellular reagents expressing Gibson assembly enzymes–Taq DNA polymerase, Taq DNA ligase, and T5 exonuclease–to assemble the new construct. (b) Cellular PCR amplification of vector and insert fragments directly from  E .  coli  bacteria bearing target DNA plasmids using 2 x 10 7  cells of Phusion cellular reagents. Assembly parts include: (i) “pATetO 6XHis full length” vector for two part assembly with Kan r  cassette bearing appropriate overlapping ends, and (ii) “pUC19 Fragments 1 and 2” for three part assembly with Kan r  cassette whose ends overlap with pUC19 vector fragments. (c) Gibson assembly of agarose gel purified and unpurified cellular PCR products using pure or cellular Gibson assembly reagents. In “negative control” samples the PCR products were incubated in Gibson reaction buffer without pure or cellular Gibson enzymes. “pATetO 6XHis + Kan r ”represents a two part Gibson assembly while “Puc19 Fragment 1 + pUC19 Fragment 2 + Kan r ” represents a three-part Gibson assembly. Representative number of clones recovered in each case in the presence of both ampicillin and kanamycin are reported.
    Figure Legend Snippet: PCR and Gibson assembly using cellular reagents. (a) Schematic depicting cellular PCR followed by cellular Gibson assembly for constructing new plasmids. Bacteria harboring target plasmids are mixed with polymerase-expressing cellular reagents and PCR is initiated by adding appropriate primers, buffer, and dNTP. The resulting PCR products are incubated with cellular reagents expressing Gibson assembly enzymes–Taq DNA polymerase, Taq DNA ligase, and T5 exonuclease–to assemble the new construct. (b) Cellular PCR amplification of vector and insert fragments directly from E . coli bacteria bearing target DNA plasmids using 2 x 10 7 cells of Phusion cellular reagents. Assembly parts include: (i) “pATetO 6XHis full length” vector for two part assembly with Kan r cassette bearing appropriate overlapping ends, and (ii) “pUC19 Fragments 1 and 2” for three part assembly with Kan r cassette whose ends overlap with pUC19 vector fragments. (c) Gibson assembly of agarose gel purified and unpurified cellular PCR products using pure or cellular Gibson assembly reagents. In “negative control” samples the PCR products were incubated in Gibson reaction buffer without pure or cellular Gibson enzymes. “pATetO 6XHis + Kan r ”represents a two part Gibson assembly while “Puc19 Fragment 1 + pUC19 Fragment 2 + Kan r ” represents a three-part Gibson assembly. Representative number of clones recovered in each case in the presence of both ampicillin and kanamycin are reported.

    Techniques Used: Polymerase Chain Reaction, Expressing, Incubation, Construct, Amplification, Plasmid Preparation, Agarose Gel Electrophoresis, Purification, Clone Assay

    29) Product Images from "Cellular reagents for diagnostics and synthetic biology"

    Article Title: Cellular reagents for diagnostics and synthetic biology

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0201681

    PCR and Gibson assembly using cellular reagents. (a) Schematic depicting cellular PCR followed by cellular Gibson assembly for constructing new plasmids. Bacteria harboring target plasmids are mixed with polymerase-expressing cellular reagents and PCR is initiated by adding appropriate primers, buffer, and dNTP. The resulting PCR products are incubated with cellular reagents expressing Gibson assembly enzymes–Taq DNA polymerase, Taq DNA ligase, and T5 exonuclease–to assemble the new construct. (b) Cellular PCR amplification of vector and insert fragments directly from  E .  coli  bacteria bearing target DNA plasmids using 2 x 10 7  cells of Phusion cellular reagents. Assembly parts include: (i) “pATetO 6XHis full length” vector for two part assembly with Kan r  cassette bearing appropriate overlapping ends, and (ii) “pUC19 Fragments 1 and 2” for three part assembly with Kan r  cassette whose ends overlap with pUC19 vector fragments. (c) Gibson assembly of agarose gel purified and unpurified cellular PCR products using pure or cellular Gibson assembly reagents. In “negative control” samples the PCR products were incubated in Gibson reaction buffer without pure or cellular Gibson enzymes. “pATetO 6XHis + Kan r ”represents a two part Gibson assembly while “Puc19 Fragment 1 + pUC19 Fragment 2 + Kan r ” represents a three-part Gibson assembly. Representative number of clones recovered in each case in the presence of both ampicillin and kanamycin are reported.
    Figure Legend Snippet: PCR and Gibson assembly using cellular reagents. (a) Schematic depicting cellular PCR followed by cellular Gibson assembly for constructing new plasmids. Bacteria harboring target plasmids are mixed with polymerase-expressing cellular reagents and PCR is initiated by adding appropriate primers, buffer, and dNTP. The resulting PCR products are incubated with cellular reagents expressing Gibson assembly enzymes–Taq DNA polymerase, Taq DNA ligase, and T5 exonuclease–to assemble the new construct. (b) Cellular PCR amplification of vector and insert fragments directly from E . coli bacteria bearing target DNA plasmids using 2 x 10 7 cells of Phusion cellular reagents. Assembly parts include: (i) “pATetO 6XHis full length” vector for two part assembly with Kan r cassette bearing appropriate overlapping ends, and (ii) “pUC19 Fragments 1 and 2” for three part assembly with Kan r cassette whose ends overlap with pUC19 vector fragments. (c) Gibson assembly of agarose gel purified and unpurified cellular PCR products using pure or cellular Gibson assembly reagents. In “negative control” samples the PCR products were incubated in Gibson reaction buffer without pure or cellular Gibson enzymes. “pATetO 6XHis + Kan r ”represents a two part Gibson assembly while “Puc19 Fragment 1 + pUC19 Fragment 2 + Kan r ” represents a three-part Gibson assembly. Representative number of clones recovered in each case in the presence of both ampicillin and kanamycin are reported.

    Techniques Used: Polymerase Chain Reaction, Expressing, Incubation, Construct, Amplification, Plasmid Preparation, Agarose Gel Electrophoresis, Purification, Clone Assay

    30) Product Images from "Chimeric Phage Nanoparticles for Rapid Characterization of Bacterial Pathogens: Detection in Complex Biological Samples and Determination of Antibiotic Sensitivity"

    Article Title: Chimeric Phage Nanoparticles for Rapid Characterization of Bacterial Pathogens: Detection in Complex Biological Samples and Determination of Antibiotic Sensitivity

    Journal: ACS Sensors

    doi: 10.1021/acssensors.0c00654

    Determination of growth in the presence of antibiotics using thiolated M13KE phage and AuNPs. (a–c) Digital photos and (d–f) UV–vis spectra are shown. Samples in (a, d), (b, e), and (c, f) were grown with ampicillin, kanamycin, or tetracycline, respectively. Samples from left to right in each photo are AuNPs with no bacteria or phages, control (10 6 CFU cells with unmodified M13KE phage and AuNPs), and thiolated M13KE phage and AuNPs with the bacterial sample at the following dilutions: 1-, 10-, 10 2 -, 10 3 -, 10 4 -, 10 5 -, 10 6 -, and 10 7 -fold.
    Figure Legend Snippet: Determination of growth in the presence of antibiotics using thiolated M13KE phage and AuNPs. (a–c) Digital photos and (d–f) UV–vis spectra are shown. Samples in (a, d), (b, e), and (c, f) were grown with ampicillin, kanamycin, or tetracycline, respectively. Samples from left to right in each photo are AuNPs with no bacteria or phages, control (10 6 CFU cells with unmodified M13KE phage and AuNPs), and thiolated M13KE phage and AuNPs with the bacterial sample at the following dilutions: 1-, 10-, 10 2 -, 10 3 -, 10 4 -, 10 5 -, 10 6 -, and 10 7 -fold.

    Techniques Used:

    31) Product Images from "Fluorescence-based methods for measuring target interference by CRISPR-Cas systems"

    Article Title: Fluorescence-based methods for measuring target interference by CRISPR-Cas systems

    Journal: Methods in enzymology

    doi: 10.1016/bs.mie.2018.10.027

    Detecting CRISPR interference in bacterial colonies. A. Design of target sequence inserted into pACYC-GFP. The perfect target is shown, similar oligonucleotides bearing G1C, A4G, AAA PAM or AGA PAM (non-target strand sequences) mutations were used for mutant target sequences. Positions of seed mutations are indicated. The target-strand protospacer is highlighted in yellow, the seed in blue, and the PAM in red. NcoI and NotI overhangs are labeled. B. Typhoon scanned plates for perfect target, empty pACYC-GFP lacking a CRISPR target, and the four mutant target plasmids. C. Box plot of quantified intensities for colonies on each plate. The mean intensity for each colony was normalized against the average mean intensity for colonies from the empty pACYC-GFP plate ([mean intensity induced colony]/[average mean intensity for all empty pACYC-GFP colonies]). Boxes depict variation from 25 th to 75 th percentile with the line within the box representing the median value and the X marking the mean. Error bars depict the local minimum and maximum, outliers are shown as circles.
    Figure Legend Snippet: Detecting CRISPR interference in bacterial colonies. A. Design of target sequence inserted into pACYC-GFP. The perfect target is shown, similar oligonucleotides bearing G1C, A4G, AAA PAM or AGA PAM (non-target strand sequences) mutations were used for mutant target sequences. Positions of seed mutations are indicated. The target-strand protospacer is highlighted in yellow, the seed in blue, and the PAM in red. NcoI and NotI overhangs are labeled. B. Typhoon scanned plates for perfect target, empty pACYC-GFP lacking a CRISPR target, and the four mutant target plasmids. C. Box plot of quantified intensities for colonies on each plate. The mean intensity for each colony was normalized against the average mean intensity for colonies from the empty pACYC-GFP plate ([mean intensity induced colony]/[average mean intensity for all empty pACYC-GFP colonies]). Boxes depict variation from 25 th to 75 th percentile with the line within the box representing the median value and the X marking the mean. Error bars depict the local minimum and maximum, outliers are shown as circles.

    Techniques Used: CRISPR, Sequencing, Mutagenesis, Labeling

    32) Product Images from "Nucleic acid evolution and minimization by nonhomologous random recombination"

    Article Title: Nucleic acid evolution and minimization by nonhomologous random recombination

    Journal: Nature biotechnology

    doi: 10.1038/nbt736

    Overview of the nonhomologous random recombination (NRR) method. (A) Starting DNA sequences are randomly digested with DNase I, blunt-ended with T4 DNA polymerase, and recombined with T4 DNA ligase under conditions that strongly favor intermolecular ligation over intramolecular circularization. (B) A defined stoichiometry of hairpin DNA added to the ligation reaction controls the average length of the recombined products. The completed ligation reaction is digested with a restriction endonuclease to provide a library of double-stranded recombined DNA flanked by defined primer-binding sequences.
    Figure Legend Snippet: Overview of the nonhomologous random recombination (NRR) method. (A) Starting DNA sequences are randomly digested with DNase I, blunt-ended with T4 DNA polymerase, and recombined with T4 DNA ligase under conditions that strongly favor intermolecular ligation over intramolecular circularization. (B) A defined stoichiometry of hairpin DNA added to the ligation reaction controls the average length of the recombined products. The completed ligation reaction is digested with a restriction endonuclease to provide a library of double-stranded recombined DNA flanked by defined primer-binding sequences.

    Techniques Used: Ligation, Binding Assay

    33) Product Images from "Impact of DNA ligase IV on the fidelity of end joining in human cells"

    Article Title: Impact of DNA ligase IV on the fidelity of end joining in human cells

    Journal: Nucleic Acids Research

    doi:

    End protection by DNA ligase IV–XRCC4 protein can occur in the absence of DNA end joining. Protein extracts (40 µg) prepared from control lymphoblastoid cell lines, AHH1 and Nalm-6, the LIG4 syndrome cell line LB2304 and the LIG4 -null cell line N114P2 were incubated with a non-ligatable 5′- 32 P-end-labeled substrate (20 ng). Recombinant DNA ligase IV–XRCC4 (180 ng) was added where shown. Product formation was analyzed by agarose gel electrophoresis followed by autoradiography.
    Figure Legend Snippet: End protection by DNA ligase IV–XRCC4 protein can occur in the absence of DNA end joining. Protein extracts (40 µg) prepared from control lymphoblastoid cell lines, AHH1 and Nalm-6, the LIG4 syndrome cell line LB2304 and the LIG4 -null cell line N114P2 were incubated with a non-ligatable 5′- 32 P-end-labeled substrate (20 ng). Recombinant DNA ligase IV–XRCC4 (180 ng) was added where shown. Product formation was analyzed by agarose gel electrophoresis followed by autoradiography.

    Techniques Used: Incubation, Labeling, Recombinant, Agarose Gel Electrophoresis, Autoradiography

    34) Product Images from "Molecular differences between two Jeryl Lynn mumps virus vaccine component strains, JL5 and JL2"

    Article Title: Molecular differences between two Jeryl Lynn mumps virus vaccine component strains, JL5 and JL2

    Journal: The Journal of General Virology

    doi: 10.1099/vir.0.013946-0

    Molecular clone of MuV JL2 , indicating gene boundaries and restriction sites in pMuV JL2 . The bar shows the antigenome of pMuV JL2 and the locations of viral genes (not to scale). Arrows beneath the bar indicate the location of unique restriction sites suitable for ligation-independent cloning using exonuclease III in pMuV JL2 . The vector sequence flanking the antigenome contains a Not I site upstream of a T7 RNA polymerase promoter located 5′ to the antigenome (i.e. to the left of N) and a Kas I site downstream of the antigenome 3′ terminus (i.e. to the right of L) which is internal to the hepatitis delta ribozyme (these restriction sites are shown in bold). (a) Restriction sites present in the consensus MuV JL2 sequence – these were either already unique in the consensus MuV JL2 sequence or made unique by mutagenesis of sites at other locations in the MuV genome or the plasmid vector. (b) Restriction sites introduced into the final clone by in vitro mutagenesis. Additional Sma I, Avr II, Bsr GI and Xho I restriction sites in the MuV JL2 sequence (c) were removed by in vitro mutagenesis. A Sap I site and two Fsp I sites were removed from the vector sequence by in vitro mutagenesis or deletion to render sites in the MuV JL2 sequence unique in the final clone. Restriction-enzyme names are abbreviated for clarity. Details of their position in the MuV JL2 sequence are available on request. The asterisks indicate that these sites are unique in the plasmid DNA which is methylated, as there are two sites at 11408–11413 and 11608–11613 that are also cleavable with Stu I and Nru I, respectively, in unmethylated plasmid DNA.
    Figure Legend Snippet: Molecular clone of MuV JL2 , indicating gene boundaries and restriction sites in pMuV JL2 . The bar shows the antigenome of pMuV JL2 and the locations of viral genes (not to scale). Arrows beneath the bar indicate the location of unique restriction sites suitable for ligation-independent cloning using exonuclease III in pMuV JL2 . The vector sequence flanking the antigenome contains a Not I site upstream of a T7 RNA polymerase promoter located 5′ to the antigenome (i.e. to the left of N) and a Kas I site downstream of the antigenome 3′ terminus (i.e. to the right of L) which is internal to the hepatitis delta ribozyme (these restriction sites are shown in bold). (a) Restriction sites present in the consensus MuV JL2 sequence – these were either already unique in the consensus MuV JL2 sequence or made unique by mutagenesis of sites at other locations in the MuV genome or the plasmid vector. (b) Restriction sites introduced into the final clone by in vitro mutagenesis. Additional Sma I, Avr II, Bsr GI and Xho I restriction sites in the MuV JL2 sequence (c) were removed by in vitro mutagenesis. A Sap I site and two Fsp I sites were removed from the vector sequence by in vitro mutagenesis or deletion to render sites in the MuV JL2 sequence unique in the final clone. Restriction-enzyme names are abbreviated for clarity. Details of their position in the MuV JL2 sequence are available on request. The asterisks indicate that these sites are unique in the plasmid DNA which is methylated, as there are two sites at 11408–11413 and 11608–11613 that are also cleavable with Stu I and Nru I, respectively, in unmethylated plasmid DNA.

    Techniques Used: Ligation, Clone Assay, Plasmid Preparation, Sequencing, Mutagenesis, In Vitro, Methylation

    35) Product Images from "Improving heterologous protein expression in Synechocystis sp. PCC 6803 for alpha-bisabolene production"

    Article Title: Improving heterologous protein expression in Synechocystis sp. PCC 6803 for alpha-bisabolene production

    Journal: Metabolic Engineering Communications

    doi: 10.1016/j.mec.2019.e00117

    Terpene synthesis in S. 6803 (A). Blue boxes indicate the heterologous genes used ispA gene from E. coli and the bisabolene synthase gene from Abies grandis . The two-step selection/counterselection transformation allows the generation of strains without selection markers (B). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
    Figure Legend Snippet: Terpene synthesis in S. 6803 (A). Blue boxes indicate the heterologous genes used ispA gene from E. coli and the bisabolene synthase gene from Abies grandis . The two-step selection/counterselection transformation allows the generation of strains without selection markers (B). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

    Techniques Used: Selection, Transformation Assay

    Comparison of bisabolene titer for five different codon optimizations of bisabolene synthase (A), and the codon adaptation index for each of these strains versus bisabolene titer (B). Error bars indicate the standard deviation of the titer measured from three biological replicates with two GC-MS technical replicates each. Asterisks indicate significant differences between a designed RBS sequence and the base case for that codon optimization with (paired t -test with p ​
    Figure Legend Snippet: Comparison of bisabolene titer for five different codon optimizations of bisabolene synthase (A), and the codon adaptation index for each of these strains versus bisabolene titer (B). Error bars indicate the standard deviation of the titer measured from three biological replicates with two GC-MS technical replicates each. Asterisks indicate significant differences between a designed RBS sequence and the base case for that codon optimization with (paired t -test with p ​

    Techniques Used: Standard Deviation, Gas Chromatography-Mass Spectrometry, Sequencing

    Bisabolene synthase relative protein abundance measured by Western blot versus bisabolene specific titer (A) and relative measured bisabolene synthase abundance versus the RBS Calculator v2.1 predicted translation initiation rate (B). Error bars represent the standard deviation the Western blot signals from three biological replicates.
    Figure Legend Snippet: Bisabolene synthase relative protein abundance measured by Western blot versus bisabolene specific titer (A) and relative measured bisabolene synthase abundance versus the RBS Calculator v2.1 predicted translation initiation rate (B). Error bars represent the standard deviation the Western blot signals from three biological replicates.

    Techniques Used: Western Blot, Standard Deviation

    36) Product Images from "Endogenous viral element-derived piRNAs are not required for production of ping-pong-dependent piRNAs from Diaphorina citri densovirus"

    Article Title: Endogenous viral element-derived piRNAs are not required for production of ping-pong-dependent piRNAs from Diaphorina citri densovirus

    Journal: bioRxiv

    doi: 10.1101/2020.05.20.105924

    Construction of CrPV-DcDV, a recombinant CrPV mutant containing 57 nt of sequence from the DcDV genome. (A) Genome organization of CrPV DcDV. Blue rectangles represent the CrPV non-structural proteins. RdRp = RNA dependent RNA polymerase. VPg = viral protein genome-linked. Green boxes represent the CrPV structural proteins. The orange rectangle represents the recombinant DcDV sequence, which corresponds to nucleotides 803-859 from the DcDV genome. Pink rectangles represent the cleavage site at which the 1A protein is released from the polyprotein. (B C) Electron micrographs of wild-type CrPV (B) or CrPV-DcDV (C) virions purified from S2 cells transfected with viral RNA. 50,000x magnification. Scale bar is 50 nm. (D) RT-PCR products produced using primers flanking the site into which recombinant DcDV sequence was inserted in CrPV-DcDV (primers 11 and 12). RNA extracted from purified virions (VR) or in vitro transcribed viral RNA (TR) was used as a template. (E-G) Bright-field microscopy images of S2 cells infected with wild-type CrPV virions (E), CrPV-DcDV virions (F), or mock infected (G). Images were acquired 72 hours post-infection.
    Figure Legend Snippet: Construction of CrPV-DcDV, a recombinant CrPV mutant containing 57 nt of sequence from the DcDV genome. (A) Genome organization of CrPV DcDV. Blue rectangles represent the CrPV non-structural proteins. RdRp = RNA dependent RNA polymerase. VPg = viral protein genome-linked. Green boxes represent the CrPV structural proteins. The orange rectangle represents the recombinant DcDV sequence, which corresponds to nucleotides 803-859 from the DcDV genome. Pink rectangles represent the cleavage site at which the 1A protein is released from the polyprotein. (B C) Electron micrographs of wild-type CrPV (B) or CrPV-DcDV (C) virions purified from S2 cells transfected with viral RNA. 50,000x magnification. Scale bar is 50 nm. (D) RT-PCR products produced using primers flanking the site into which recombinant DcDV sequence was inserted in CrPV-DcDV (primers 11 and 12). RNA extracted from purified virions (VR) or in vitro transcribed viral RNA (TR) was used as a template. (E-G) Bright-field microscopy images of S2 cells infected with wild-type CrPV virions (E), CrPV-DcDV virions (F), or mock infected (G). Images were acquired 72 hours post-infection.

    Techniques Used: Recombinant, Mutagenesis, Sequencing, Purification, Transfection, Reverse Transcription Polymerase Chain Reaction, Produced, In Vitro, Microscopy, Infection

    Alignment of a portion of the CrPV-DcDV genome, the recombinant DcDV sequence present within CrPV-DcDV, the region of ENS corresponding to the recombinant DcDV sequence present within CrPV-DcDV (represented by the GenBank accession no. of D. citri genomic scaffold 2850, NW_007380266), and a portion of the wild-type CrPV sequence. Numbers in parentheses indicate the nucleotide positions of the sequence shown. The CrPV 1A cleavage site is shown in lowercase letters. The recombinant DcDV sequence present within CrPV-DcDV and the corresponding region of ENS are shown in italics.
    Figure Legend Snippet: Alignment of a portion of the CrPV-DcDV genome, the recombinant DcDV sequence present within CrPV-DcDV, the region of ENS corresponding to the recombinant DcDV sequence present within CrPV-DcDV (represented by the GenBank accession no. of D. citri genomic scaffold 2850, NW_007380266), and a portion of the wild-type CrPV sequence. Numbers in parentheses indicate the nucleotide positions of the sequence shown. The CrPV 1A cleavage site is shown in lowercase letters. The recombinant DcDV sequence present within CrPV-DcDV and the corresponding region of ENS are shown in italics.

    Techniques Used: Recombinant, Sequencing

    A DcDV-like EVE is present in the D. citri genome. (A) Organization of DcDV-derived EVEs identified in D. citri genomic scaffold 2850 by BLASTx followed by manual sequence alignment. The DcDV genome organization is shown on top with the corresponding region of scaffold 2850 on the bottom. Numbers above and below the sequence depictions represent nucleotide positions. Vertical lines inside shaded boxes represent stop codons. The percent nucleotide or deduced amino acid (aa) identity between EVEs and corresponding viral genomic regions/proteins is given. Annealing positions of PCR primers are shown with arrows. (B) Confirmation of EVE presence by PCR using primers shown in panel A. The product produced with primers 5/6 was produced by nested PCR using as template a 1:1000 dilution of a PCR product produced with primers 3/4. (C) The correct organization of the region of the D. citri genome containing ENS and EITR, as confirmed by sequencing of the PCR products shown in panel B.
    Figure Legend Snippet: A DcDV-like EVE is present in the D. citri genome. (A) Organization of DcDV-derived EVEs identified in D. citri genomic scaffold 2850 by BLASTx followed by manual sequence alignment. The DcDV genome organization is shown on top with the corresponding region of scaffold 2850 on the bottom. Numbers above and below the sequence depictions represent nucleotide positions. Vertical lines inside shaded boxes represent stop codons. The percent nucleotide or deduced amino acid (aa) identity between EVEs and corresponding viral genomic regions/proteins is given. Annealing positions of PCR primers are shown with arrows. (B) Confirmation of EVE presence by PCR using primers shown in panel A. The product produced with primers 5/6 was produced by nested PCR using as template a 1:1000 dilution of a PCR product produced with primers 3/4. (C) The correct organization of the region of the D. citri genome containing ENS and EITR, as confirmed by sequencing of the PCR products shown in panel B.

    Techniques Used: Derivative Assay, Sequencing, Polymerase Chain Reaction, Produced, Nested PCR

    RT-PCR products produced using primers flanking the site into which recombinant DcDV sequence was inserted in CrPV-DcDV (primers 15 and 16). RNA from all biological replicates from each day shown in Fig. 4.6a (A) or Fig. 4.7a (B) was pooled and used as templates for RT-PCR. RNA from CRF-CA D. citri was used as a negative control. In vitro transcribed wild-type CrPV or CrPV-DcDV RNA was used as a positive control. DPI = days post injection. DPF = days post feeding.
    Figure Legend Snippet: RT-PCR products produced using primers flanking the site into which recombinant DcDV sequence was inserted in CrPV-DcDV (primers 15 and 16). RNA from all biological replicates from each day shown in Fig. 4.6a (A) or Fig. 4.7a (B) was pooled and used as templates for RT-PCR. RNA from CRF-CA D. citri was used as a negative control. In vitro transcribed wild-type CrPV or CrPV-DcDV RNA was used as a positive control. DPI = days post injection. DPF = days post feeding.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Produced, Recombinant, Sequencing, Negative Control, In Vitro, Positive Control, Injection

    37) Product Images from "DNA supercoiling, a critical signal regulating the basal expression of the lac operon in Escherichia coli"

    Article Title: DNA supercoiling, a critical signal regulating the basal expression of the lac operon in Escherichia coli

    Journal: Scientific Reports

    doi: 10.1038/srep19243

    One molecule of LacI tetramer divided a supercoiled DNA molecule plasmid pCB126 into two independent topological domains. ( a ) Plasmid pCB126 carrying two lac O1 operators in two different locations was constructed as detailed in Methods. ( b ) The nicking enzyme Nt.BbvCI was able to rapidly digest pCB126. Time course of enzyme digestion of pCB126 using 16 units of Nt.BbvCI in 1 × NEBuffer 4 at 37 °C. Lane 1 contained the undigested scDNA. ( c ) Time course of DNA supercoiling diffusion in the presence of LacI. The DNA-nicking assays were performed as described under Methods. Each reaction mixture (320 μL) contained 0.156 nM of pCB126, 2.5 nM of LacI, and 16 units of Nt.BbvCI. The reactions were incubated at 37 °C for the time indicated. Then a large excess of a double-stranded oligonucleotide contain an Nt.BbvCI recognition site was added to the reaction mixture to inhibit the restriction enzyme activities. The nicked DNA templates were ligated by T4 DNA ligase in the presence of 1 mM of ATP at 37 °C for 5 min and the reactions were terminated by phenol extraction. The DNA molecules were isolated and subjected to agarose gel electrophoresis. ( d ) Quantification analysis of the time course. The percentage of supercoiled DNA was plotted against the reaction time. The curve was generated by fitting the data to a 1st-order rate equation to yield a rate constant of 0.016 sec −1 and a t 1/2 of 52 sec.
    Figure Legend Snippet: One molecule of LacI tetramer divided a supercoiled DNA molecule plasmid pCB126 into two independent topological domains. ( a ) Plasmid pCB126 carrying two lac O1 operators in two different locations was constructed as detailed in Methods. ( b ) The nicking enzyme Nt.BbvCI was able to rapidly digest pCB126. Time course of enzyme digestion of pCB126 using 16 units of Nt.BbvCI in 1 × NEBuffer 4 at 37 °C. Lane 1 contained the undigested scDNA. ( c ) Time course of DNA supercoiling diffusion in the presence of LacI. The DNA-nicking assays were performed as described under Methods. Each reaction mixture (320 μL) contained 0.156 nM of pCB126, 2.5 nM of LacI, and 16 units of Nt.BbvCI. The reactions were incubated at 37 °C for the time indicated. Then a large excess of a double-stranded oligonucleotide contain an Nt.BbvCI recognition site was added to the reaction mixture to inhibit the restriction enzyme activities. The nicked DNA templates were ligated by T4 DNA ligase in the presence of 1 mM of ATP at 37 °C for 5 min and the reactions were terminated by phenol extraction. The DNA molecules were isolated and subjected to agarose gel electrophoresis. ( d ) Quantification analysis of the time course. The percentage of supercoiled DNA was plotted against the reaction time. The curve was generated by fitting the data to a 1st-order rate equation to yield a rate constant of 0.016 sec −1 and a t 1/2 of 52 sec.

    Techniques Used: Plasmid Preparation, Construct, Diffusion-based Assay, Incubation, Isolation, Agarose Gel Electrophoresis, Generated, Size-exclusion Chromatography

    38) Product Images from "Biotechnology by Design: An Introductory Level, Project-Based, Synthetic Biology Laboratory Program for Undergraduate Students †"

    Article Title: Biotechnology by Design: An Introductory Level, Project-Based, Synthetic Biology Laboratory Program for Undergraduate Students †

    Journal: Journal of Microbiology & Biology Education

    doi: 10.1128/jmbe.v16i2.971

    BioBrick Assembly Methods Flowchart. The flowchart illustrates the stepwise construction of the reporter plasmid using a Back-End Assembly approach. Molecular biology techniques are listed in blue type, and decision points are outlined in red. Verification
    Figure Legend Snippet: BioBrick Assembly Methods Flowchart. The flowchart illustrates the stepwise construction of the reporter plasmid using a Back-End Assembly approach. Molecular biology techniques are listed in blue type, and decision points are outlined in red. Verification

    Techniques Used: Plasmid Preparation

    39) Product Images from "Efficient expression of codon-adapted affinity tagged super folder green fluorescent protein for synchronous protein localization and affinity purification studies in Tetrahymena thermophila"

    Article Title: Efficient expression of codon-adapted affinity tagged super folder green fluorescent protein for synchronous protein localization and affinity purification studies in Tetrahymena thermophila

    Journal: BMC Biotechnology

    doi: 10.1186/s12896-015-0137-9

    Comparative in vivo fluorescent spectrophotometric analyses of cells expressing EGFP, TtsfGFP, and sfGFP. Tetrahymena clones carrying TtsfGFP-, sfGFP-, and EGFP-expressing constructs were grown until the mid-logarithmic phase and cell density adjusted to 3 × 105 cells/mL. The cells were induced with 2 μg/mL of CdCl2. In vivo fluorescence spectrophotometric analyses were performed every 30 min at 488 nm excitation and 510 nm emission. The difference in emission between TtsfGFP, sfGFP, and EGFP began at 60 min and continued until 180 min. At the end of the time interval, TtsfGFP clones were found to emit ~2.2 fold and ~4 fold more fluorescence than sfGFP and EGFP, respectively.
    Figure Legend Snippet: Comparative in vivo fluorescent spectrophotometric analyses of cells expressing EGFP, TtsfGFP, and sfGFP. Tetrahymena clones carrying TtsfGFP-, sfGFP-, and EGFP-expressing constructs were grown until the mid-logarithmic phase and cell density adjusted to 3 × 105 cells/mL. The cells were induced with 2 μg/mL of CdCl2. In vivo fluorescence spectrophotometric analyses were performed every 30 min at 488 nm excitation and 510 nm emission. The difference in emission between TtsfGFP, sfGFP, and EGFP began at 60 min and continued until 180 min. At the end of the time interval, TtsfGFP clones were found to emit ~2.2 fold and ~4 fold more fluorescence than sfGFP and EGFP, respectively.

    Techniques Used: In Vivo, Expressing, Clone Assay, Construct, Fluorescence

    Constructed protein expression vectors of T. thermophila . A. Vector pVTtsfGFP includes the T. thermophila codon-adapted TtsfGFP expression cassette; B. Vector pVsfGFP carries a non-codon-adapted sfGFP expression cassette; C. Vector pEGFP includes the non-codon-adapted EGFP expression cassette. All vectors were derived from pVGF. MTT1 is a cadmium-inducible promotor. The transcription is terminated using an rpL29 termination sequence.
    Figure Legend Snippet: Constructed protein expression vectors of T. thermophila . A. Vector pVTtsfGFP includes the T. thermophila codon-adapted TtsfGFP expression cassette; B. Vector pVsfGFP carries a non-codon-adapted sfGFP expression cassette; C. Vector pEGFP includes the non-codon-adapted EGFP expression cassette. All vectors were derived from pVGF. MTT1 is a cadmium-inducible promotor. The transcription is terminated using an rpL29 termination sequence.

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

    Western blot analysis of 6 × His-TtsfGFP purified with by a Ni-NTA column. The purified 6 × His-TtsfGFP isolated from cells with 2 μg/mL of CdCl 2 for 18 h. From the Ni-NTA affinity purification columns, the recombinant 6 × His-TtsfGFP was observed as ~34 kDa protein in the first elution (Lane 1, arrow), the flow-through (Lane 2), and the first wash (Lane 3) determined using a monoclonal mouse anti-GFP antibody as the primary antibody(1:1000). M: Bio-Rad Kaleidoscope western markers.
    Figure Legend Snippet: Western blot analysis of 6 × His-TtsfGFP purified with by a Ni-NTA column. The purified 6 × His-TtsfGFP isolated from cells with 2 μg/mL of CdCl 2 for 18 h. From the Ni-NTA affinity purification columns, the recombinant 6 × His-TtsfGFP was observed as ~34 kDa protein in the first elution (Lane 1, arrow), the flow-through (Lane 2), and the first wash (Lane 3) determined using a monoclonal mouse anti-GFP antibody as the primary antibody(1:1000). M: Bio-Rad Kaleidoscope western markers.

    Techniques Used: Western Blot, Purification, Isolation, Affinity Purification, Recombinant, Flow Cytometry

    Fluorescent and western blot analysis of the hypothetical ATP dependent DNA ligase domain containing protein. A. SDS-PAGE was carried out in a discontinuous slab gel under semi-denaturing conditions by omitting mercaptoethanol from the sample buffer and without boiling. About 15 μg of total protein were loaded in each lane. Gels were visualized with Bio-Rad Gel Doc EZ using the blue sample tray for GFP. Lane 1: affinity purified 6 × His-TtsfGFP from Eschericha coli ; lane 2: untransformed T. thermophila total cell protein (negative control); T. thermophila with pVTtsfGFP-H induced with 0.25 μg/mL of CdCl 2 for 3 h; lane 3: zero time, lane 4: 1 h, lane 5: 2 h, lane 6: 3 h. B. The 6 × His-TtsfGFP-H fusion protein purified from T. thermophila pVTtsfGFP-H clone was induced for 18 h and analyzed with western blotting by using monoclonal mouse anti-GFP antibody (1:1000). The 6 × His-TtsfGFP-H was approximately 95 kDa (Lane 1, black arrow), as expected. Many fragmented proteins were also visible. Moreover, some of the target and fragmented fusion proteins were lost during washing (Lane 2) and flow-through (Lane 3) steps of Ni-NTA affinity purification. The predicted size of the fragments based on the rare codons plus 6 × His-TtsfGFP would be approximately 36.7 kDa (Arrow 1), 48 kDa (Arrow 2) and 50 kDa (Arrow 3). The roughly 70 kDa band could be dimer of these broken fusion proteins caused by the dimerization of sfGFP (Arrow 4). M: Bio-Rad Kaleidoscope western marker.
    Figure Legend Snippet: Fluorescent and western blot analysis of the hypothetical ATP dependent DNA ligase domain containing protein. A. SDS-PAGE was carried out in a discontinuous slab gel under semi-denaturing conditions by omitting mercaptoethanol from the sample buffer and without boiling. About 15 μg of total protein were loaded in each lane. Gels were visualized with Bio-Rad Gel Doc EZ using the blue sample tray for GFP. Lane 1: affinity purified 6 × His-TtsfGFP from Eschericha coli ; lane 2: untransformed T. thermophila total cell protein (negative control); T. thermophila with pVTtsfGFP-H induced with 0.25 μg/mL of CdCl 2 for 3 h; lane 3: zero time, lane 4: 1 h, lane 5: 2 h, lane 6: 3 h. B. The 6 × His-TtsfGFP-H fusion protein purified from T. thermophila pVTtsfGFP-H clone was induced for 18 h and analyzed with western blotting by using monoclonal mouse anti-GFP antibody (1:1000). The 6 × His-TtsfGFP-H was approximately 95 kDa (Lane 1, black arrow), as expected. Many fragmented proteins were also visible. Moreover, some of the target and fragmented fusion proteins were lost during washing (Lane 2) and flow-through (Lane 3) steps of Ni-NTA affinity purification. The predicted size of the fragments based on the rare codons plus 6 × His-TtsfGFP would be approximately 36.7 kDa (Arrow 1), 48 kDa (Arrow 2) and 50 kDa (Arrow 3). The roughly 70 kDa band could be dimer of these broken fusion proteins caused by the dimerization of sfGFP (Arrow 4). M: Bio-Rad Kaleidoscope western marker.

    Techniques Used: Western Blot, SDS Page, Affinity Purification, Negative Control, Purification, Flow Cytometry, Marker

    Comparative in vivo fluorescence microscope analyses of EGFP, TtsfGFP, sfGFP expression. The TtsfGFP-expressing cells emitted brighter green fluorescent light than the sfGFP and EGFP expressing clones at all period after induction with 2 μg/mL of CdCl 2 . A Leica DM6000 B fluorescence microscope equipped with a 20× objective and a GFP filter was used. Photo revision using PhotoScape 3.6.3 software was performed without any misleading changes because of the low quality of the images.
    Figure Legend Snippet: Comparative in vivo fluorescence microscope analyses of EGFP, TtsfGFP, sfGFP expression. The TtsfGFP-expressing cells emitted brighter green fluorescent light than the sfGFP and EGFP expressing clones at all period after induction with 2 μg/mL of CdCl 2 . A Leica DM6000 B fluorescence microscope equipped with a 20× objective and a GFP filter was used. Photo revision using PhotoScape 3.6.3 software was performed without any misleading changes because of the low quality of the images.

    Techniques Used: In Vivo, Fluorescence, Microscopy, Expressing, Clone Assay, Software

    Western blot analysis of total proteins from T. thermophila cells expressing TtsfGFP, sfGFP, or EGFP. Equal amounts (30 μg) of EGFP (Lane 1), TtsfGFP (Lane 2), and sfGFP (Lane 3) were loaded. The left arrow shows a molecular mass of around 34 kDa corresponding to TtsfGFP. The quantity of TtsfGFP in the total protein extract appeared to be approximately 5–10-fold higher than sfGFP and EGFP. Total protein extracted from a Tetrahymena B2086 and CU428 cell mixture was used as a negative control (Lane 4). The positive control was TtsfGFP, which was constructed, expressed, and purified using Ni-NTA affinity purification from E. coli . The ~68 kDa band was predicted to be an sfGFP dimer (right arrow). Western blotting was performed with a monoclonal mouse anti-GFP antibody (1:1000). M: Bio-Rad Kaleidoscope western markers.
    Figure Legend Snippet: Western blot analysis of total proteins from T. thermophila cells expressing TtsfGFP, sfGFP, or EGFP. Equal amounts (30 μg) of EGFP (Lane 1), TtsfGFP (Lane 2), and sfGFP (Lane 3) were loaded. The left arrow shows a molecular mass of around 34 kDa corresponding to TtsfGFP. The quantity of TtsfGFP in the total protein extract appeared to be approximately 5–10-fold higher than sfGFP and EGFP. Total protein extracted from a Tetrahymena B2086 and CU428 cell mixture was used as a negative control (Lane 4). The positive control was TtsfGFP, which was constructed, expressed, and purified using Ni-NTA affinity purification from E. coli . The ~68 kDa band was predicted to be an sfGFP dimer (right arrow). Western blotting was performed with a monoclonal mouse anti-GFP antibody (1:1000). M: Bio-Rad Kaleidoscope western markers.

    Techniques Used: Western Blot, Expressing, Negative Control, Positive Control, Construct, Purification, Affinity Purification

    Comparison of the DNA sequence of codon-optimized TtsfGFP with that of sfGFP. Top DNA sequence is the codon-adapted TtsfGFP and the bottom sequence is that of sfGFP. The 126 mutated bases are shown in bold letters. There was no change in the amino acid sequence of sfGFP.
    Figure Legend Snippet: Comparison of the DNA sequence of codon-optimized TtsfGFP with that of sfGFP. Top DNA sequence is the codon-adapted TtsfGFP and the bottom sequence is that of sfGFP. The 126 mutated bases are shown in bold letters. There was no change in the amino acid sequence of sfGFP.

    Techniques Used: Sequencing

    The macronuclear and micronuclear localization of TtsfGFP-H fusion protein in T. thermophila . pVTtsfGFP carrying T. thermophila cells as the control and T. thermophila carrying pVTtsfGFP-H cells were induced with 0.25 μg/ml of CdCl 2 for one hour. 6 × His-TtsfGFP-H was localized to the macro- (arrowed large structure) and micronucleus (arrowed small structure). However, there was no detectable localization of the 6 × His-TtsfGFP tag protein except in the cytoplasm. The images were taken by a Leica DM6000 B fluorescence microscope equipped with a 63× objective. GFP filter was used for TtsfGFP (last column) and A filter for DAPI/Hoechst 33258 staining (middle column). The first picture columns were taken with light microscopy. The 2–3 vesicles that are seen in the cytoplasm are unknown structures.
    Figure Legend Snippet: The macronuclear and micronuclear localization of TtsfGFP-H fusion protein in T. thermophila . pVTtsfGFP carrying T. thermophila cells as the control and T. thermophila carrying pVTtsfGFP-H cells were induced with 0.25 μg/ml of CdCl 2 for one hour. 6 × His-TtsfGFP-H was localized to the macro- (arrowed large structure) and micronucleus (arrowed small structure). However, there was no detectable localization of the 6 × His-TtsfGFP tag protein except in the cytoplasm. The images were taken by a Leica DM6000 B fluorescence microscope equipped with a 63× objective. GFP filter was used for TtsfGFP (last column) and A filter for DAPI/Hoechst 33258 staining (middle column). The first picture columns were taken with light microscopy. The 2–3 vesicles that are seen in the cytoplasm are unknown structures.

    Techniques Used: Fluorescence, Microscopy, Staining, Light Microscopy

    40) Product Images from "Generation and analysis of a barcode-tagged insertion mutant library in the fission yeast Schizosaccharomyces pombe"

    Article Title: Generation and analysis of a barcode-tagged insertion mutant library in the fission yeast Schizosaccharomyces pombe

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-13-161

    Ligation-mediated barcode oligomerization. ( A ) Barcodes in this insertion mutant library can be amplified from a population of mutants by PCR using vector-specific primers that flank barcodes. Overhangs generated by Sfi I digestion allow barcode monomers to be oligomerized in a head-to-tail manner. ( B ) A representative barcode oligomerization. The lanes labeled “monomer” and “oligomers” show the Sfi I-digested barcode DNA before and after ligation, respectively.
    Figure Legend Snippet: Ligation-mediated barcode oligomerization. ( A ) Barcodes in this insertion mutant library can be amplified from a population of mutants by PCR using vector-specific primers that flank barcodes. Overhangs generated by Sfi I digestion allow barcode monomers to be oligomerized in a head-to-tail manner. ( B ) A representative barcode oligomerization. The lanes labeled “monomer” and “oligomers” show the Sfi I-digested barcode DNA before and after ligation, respectively.

    Techniques Used: Ligation, Mutagenesis, Amplification, Polymerase Chain Reaction, Plasmid Preparation, Generated, Labeling

    A non-homologous recombination-mediated insertion mutagenesis for generating an S. pombe mutant library. ( A ) The first insertion vector tested had the selectable marker ura4 + and a 15-bp random barcode directly following the 3’ UTR of ura4 + . ( B ) The insertion vector used to construct the S. pombe insertion mutant library is composed of a selectable marker ura4 + gene, a barcode (27 random nucleotides with 14 interspersed A’s), a lox71 site for one-way integration of lox66-bearing DNA, a mutated human HSP70 promoter with a lexA binding site and a modified λ phage sequence, ATG-less λ, to protect the sequences 3’ to the λ phage fragment from degradation.
    Figure Legend Snippet: A non-homologous recombination-mediated insertion mutagenesis for generating an S. pombe mutant library. ( A ) The first insertion vector tested had the selectable marker ura4 + and a 15-bp random barcode directly following the 3’ UTR of ura4 + . ( B ) The insertion vector used to construct the S. pombe insertion mutant library is composed of a selectable marker ura4 + gene, a barcode (27 random nucleotides with 14 interspersed A’s), a lox71 site for one-way integration of lox66-bearing DNA, a mutated human HSP70 promoter with a lexA binding site and a modified λ phage sequence, ATG-less λ, to protect the sequences 3’ to the λ phage fragment from degradation.

    Techniques Used: Homologous Recombination, Mutagenesis, Plasmid Preparation, Marker, Construct, Binding Assay, Modification, Sequencing

    Generation of the barcode-tagged S. pombe insertion mutant library. The linear insertion DNA (Figure 1 B) was transformed into the wild type strain KRP1 to obtain Ura + transformants on minimal medium (MMA) with multiple nutritional supplements except uracil (YC – uracil) and low levels of 5-FOA (0.1 g/l). Transformants were then tested for stable integration by 5-FOA sensitivity. Stable transformants (i.e. 5-FOA sensitive cells) were inoculated in non-selective YES medium in 96-well plates, followed by assembling four such plates on a synthetic medium plate lacking uracil (EMM + YC – uracil) and a similar medium plate that contains uracil and 1 g/l of 5-FOA (EMM + YC + 5-FOA) to generate 384-colony arrays for the second 5-FOA sensitivity test. Unstable transformants found in this second screen were removed before these mutants were stored as 384-well mutant arrays or mixed mutant pools of ~1800 mutants.
    Figure Legend Snippet: Generation of the barcode-tagged S. pombe insertion mutant library. The linear insertion DNA (Figure 1 B) was transformed into the wild type strain KRP1 to obtain Ura + transformants on minimal medium (MMA) with multiple nutritional supplements except uracil (YC – uracil) and low levels of 5-FOA (0.1 g/l). Transformants were then tested for stable integration by 5-FOA sensitivity. Stable transformants (i.e. 5-FOA sensitive cells) were inoculated in non-selective YES medium in 96-well plates, followed by assembling four such plates on a synthetic medium plate lacking uracil (EMM + YC – uracil) and a similar medium plate that contains uracil and 1 g/l of 5-FOA (EMM + YC + 5-FOA) to generate 384-colony arrays for the second 5-FOA sensitivity test. Unstable transformants found in this second screen were removed before these mutants were stored as 384-well mutant arrays or mixed mutant pools of ~1800 mutants.

    Techniques Used: Mutagenesis, Transformation Assay

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    Molecular clone of MuV JL2 , indicating gene boundaries and restriction sites in pMuV JL2 . The bar shows the antigenome of pMuV JL2 and the locations of viral genes (not to scale). Arrows beneath the bar indicate the location of unique restriction sites suitable for ligation-independent cloning using exonuclease <t>III</t> in pMuV JL2 . The vector sequence flanking the antigenome contains a Not I site upstream of a T7 RNA polymerase promoter located 5′ to the antigenome (i.e. to the left of N) and a Kas I site downstream of the antigenome 3′ terminus (i.e. to the right of L) which is internal to the hepatitis delta ribozyme (these restriction sites are shown in bold). (a) Restriction sites present in the consensus MuV JL2 sequence – these were either already unique in the consensus MuV JL2 sequence or made unique by mutagenesis of sites at other locations in the MuV genome or the plasmid vector. (b) Restriction sites introduced into the final clone by in vitro mutagenesis. Additional Sma I, Avr II, Bsr GI and Xho I restriction sites in the MuV JL2 sequence (c) were removed by in vitro mutagenesis. A Sap I site and two Fsp I sites were removed from the vector sequence by in vitro mutagenesis or deletion to render sites in the MuV JL2 sequence unique in the final clone. Restriction-enzyme names are abbreviated for clarity. Details of their position in the MuV JL2 sequence are available on request. The asterisks indicate that these sites are unique in the plasmid <t>DNA</t> which is methylated, as there are two sites at 11408–11413 and 11608–11613 that are also cleavable with Stu I and Nru I, respectively, in unmethylated plasmid DNA.
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    Molecular clone of MuV JL2 , indicating gene boundaries and restriction sites in pMuV JL2 . The bar shows the antigenome of pMuV JL2 and the locations of viral genes (not to scale). Arrows beneath the bar indicate the location of unique restriction sites suitable for ligation-independent cloning using exonuclease III in pMuV JL2 . The vector sequence flanking the antigenome contains a Not I site upstream of a T7 RNA polymerase promoter located 5′ to the antigenome (i.e. to the left of N) and a Kas I site downstream of the antigenome 3′ terminus (i.e. to the right of L) which is internal to the hepatitis delta ribozyme (these restriction sites are shown in bold). (a) Restriction sites present in the consensus MuV JL2 sequence – these were either already unique in the consensus MuV JL2 sequence or made unique by mutagenesis of sites at other locations in the MuV genome or the plasmid vector. (b) Restriction sites introduced into the final clone by in vitro mutagenesis. Additional Sma I, Avr II, Bsr GI and Xho I restriction sites in the MuV JL2 sequence (c) were removed by in vitro mutagenesis. A Sap I site and two Fsp I sites were removed from the vector sequence by in vitro mutagenesis or deletion to render sites in the MuV JL2 sequence unique in the final clone. Restriction-enzyme names are abbreviated for clarity. Details of their position in the MuV JL2 sequence are available on request. The asterisks indicate that these sites are unique in the plasmid DNA which is methylated, as there are two sites at 11408–11413 and 11608–11613 that are also cleavable with Stu I and Nru I, respectively, in unmethylated plasmid DNA.

    Journal: The Journal of General Virology

    Article Title: Molecular differences between two Jeryl Lynn mumps virus vaccine component strains, JL5 and JL2

    doi: 10.1099/vir.0.013946-0

    Figure Lengend Snippet: Molecular clone of MuV JL2 , indicating gene boundaries and restriction sites in pMuV JL2 . The bar shows the antigenome of pMuV JL2 and the locations of viral genes (not to scale). Arrows beneath the bar indicate the location of unique restriction sites suitable for ligation-independent cloning using exonuclease III in pMuV JL2 . The vector sequence flanking the antigenome contains a Not I site upstream of a T7 RNA polymerase promoter located 5′ to the antigenome (i.e. to the left of N) and a Kas I site downstream of the antigenome 3′ terminus (i.e. to the right of L) which is internal to the hepatitis delta ribozyme (these restriction sites are shown in bold). (a) Restriction sites present in the consensus MuV JL2 sequence – these were either already unique in the consensus MuV JL2 sequence or made unique by mutagenesis of sites at other locations in the MuV genome or the plasmid vector. (b) Restriction sites introduced into the final clone by in vitro mutagenesis. Additional Sma I, Avr II, Bsr GI and Xho I restriction sites in the MuV JL2 sequence (c) were removed by in vitro mutagenesis. A Sap I site and two Fsp I sites were removed from the vector sequence by in vitro mutagenesis or deletion to render sites in the MuV JL2 sequence unique in the final clone. Restriction-enzyme names are abbreviated for clarity. Details of their position in the MuV JL2 sequence are available on request. The asterisks indicate that these sites are unique in the plasmid DNA which is methylated, as there are two sites at 11408–11413 and 11608–11613 that are also cleavable with Stu I and Nru I, respectively, in unmethylated plasmid DNA.

    Article Snippet: Restriction enzymes, reverse transcriptase SuperScript III, high-fidelity Taq DNA polymerase, Pfu polymerase, Phusion DNA polymerase, Klenow fragment of DNA polymerase, exonuclease III and DNA ligase were obtained from New England Biolabs (NEB) or Invitrogen and used according to the manufacturers' instructions.

    Techniques: Ligation, Clone Assay, Plasmid Preparation, Sequencing, Mutagenesis, In Vitro, Methylation

    In vitro construction of ADP fragment using overlap-extension PCR. ADP fragment was amplified by PCR through 32 cycles include denaturing step (95 °C for 45 s), annealing step (60 °C for 45 s) and elongation step (72 °C for 1 min). The two fragments were then purified and joined through 10 cycles of overlap-extension PCR include denaturing step at 95 °C for 45 s, annealing step at 60 °C for 45 s and elongating step at 72 °C for 1 min. L1 : PCR product of exon 2 (204 bp). L2 : PCR product of exon 3 (531 bp). L3 : the full length of ADP fragment (734 bp). M : 100 bp DNA marker.

    Journal: International Journal of Molecular Sciences

    Article Title: A Comparative Study on the Expression, Purification and Functional Characterization of Human Adiponectin in Pichia pastoris and Escherichia coli

    doi: 10.3390/ijms13033549

    Figure Lengend Snippet: In vitro construction of ADP fragment using overlap-extension PCR. ADP fragment was amplified by PCR through 32 cycles include denaturing step (95 °C for 45 s), annealing step (60 °C for 45 s) and elongation step (72 °C for 1 min). The two fragments were then purified and joined through 10 cycles of overlap-extension PCR include denaturing step at 95 °C for 45 s, annealing step at 60 °C for 45 s and elongating step at 72 °C for 1 min. L1 : PCR product of exon 2 (204 bp). L2 : PCR product of exon 3 (531 bp). L3 : the full length of ADP fragment (734 bp). M : 100 bp DNA marker.

    Article Snippet: The ligation mixture was prepared by adding digested vector and digested ADP fragment with DNA ligase and its suitable ligation buffer (New England Biolabs, UK).

    Techniques: In Vitro, Polymerase Chain Reaction, Amplification, Purification, Marker

    Analysis of supercoiling of relaxed pHOT1 by E. coli gyrase in the presence of DMA. Lane 1, relaxed DNA as control; lane 2, supercoiling of relaxed DNA by E. coli DNA gyrase; lane 3; relaxed DNA in the presence of 100 μM DMA; lanes 4–10,

    Journal: Journal of Antimicrobial Chemotherapy

    Article Title: 3,4-Dimethoxyphenyl bis-benzimidazole, a novel DNA topoisomerase inhibitor that preferentially targets Escherichia coli topoisomerase I

    doi: 10.1093/jac/dks322

    Figure Lengend Snippet: Analysis of supercoiling of relaxed pHOT1 by E. coli gyrase in the presence of DMA. Lane 1, relaxed DNA as control; lane 2, supercoiling of relaxed DNA by E. coli DNA gyrase; lane 3; relaxed DNA in the presence of 100 μM DMA; lanes 4–10,

    Article Snippet: E. coli DNA gyrase and its relaxed substrate were purchased from New England Biolabs (GmBH, Germany).

    Techniques:

    Decreased E. coli gyrase binding to DAP DNA. Equal amounts of normal or DAP DNA were incubated with increasing E. coli ). Average percentages of binding to gyrase at different

    Journal: Journal of molecular biology

    Article Title: E. coli gyrase fails to negatively supercoil diaminopurine-substituted DNA

    doi: 10.1016/j.jmb.2015.04.006

    Figure Lengend Snippet: Decreased E. coli gyrase binding to DAP DNA. Equal amounts of normal or DAP DNA were incubated with increasing E. coli ). Average percentages of binding to gyrase at different

    Article Snippet: E. coli DNA gyrase (NEB) was used at a final concentration of about 9.2 nM (20 units/ml).

    Techniques: Binding Assay, Incubation

    Decreased gyrase wrapping of DAP DNA. (a) A schematic of the equilibrium between wrapped and unwrapped states of gyrase with no ATP present shows that the extension of the DNA tether switches between two levels. (b and c) Raw data (dots) and 10 second

    Journal: Journal of molecular biology

    Article Title: E. coli gyrase fails to negatively supercoil diaminopurine-substituted DNA

    doi: 10.1016/j.jmb.2015.04.006

    Figure Lengend Snippet: Decreased gyrase wrapping of DAP DNA. (a) A schematic of the equilibrium between wrapped and unwrapped states of gyrase with no ATP present shows that the extension of the DNA tether switches between two levels. (b and c) Raw data (dots) and 10 second

    Article Snippet: E. coli DNA gyrase (NEB) was used at a final concentration of about 9.2 nM (20 units/ml).

    Techniques:

    The lifetimes of the wrapped states of gyrase for DAP (red circles) and normal (blue crosses) DNA under 0.4 pN of tension were measured and the fraction greater than or equal to different time intervals was plotted. The number of measured pauses was 154

    Journal: Journal of molecular biology

    Article Title: E. coli gyrase fails to negatively supercoil diaminopurine-substituted DNA

    doi: 10.1016/j.jmb.2015.04.006

    Figure Lengend Snippet: The lifetimes of the wrapped states of gyrase for DAP (red circles) and normal (blue crosses) DNA under 0.4 pN of tension were measured and the fraction greater than or equal to different time intervals was plotted. The number of measured pauses was 154

    Article Snippet: E. coli DNA gyrase (NEB) was used at a final concentration of about 9.2 nM (20 units/ml).

    Techniques:

    Gyrase relaxes DAP DNA more slowly. (a) Three offset, representative records of gyrase-catalyzed relaxations of normal (left) and DAP DNA (right) under 0.6 pN tension and with 1 mM ATP. The raw data (dots) and 0.5 s moving averages (solid lines) show

    Journal: Journal of molecular biology

    Article Title: E. coli gyrase fails to negatively supercoil diaminopurine-substituted DNA

    doi: 10.1016/j.jmb.2015.04.006

    Figure Lengend Snippet: Gyrase relaxes DAP DNA more slowly. (a) Three offset, representative records of gyrase-catalyzed relaxations of normal (left) and DAP DNA (right) under 0.6 pN tension and with 1 mM ATP. The raw data (dots) and 0.5 s moving averages (solid lines) show

    Article Snippet: E. coli DNA gyrase (NEB) was used at a final concentration of about 9.2 nM (20 units/ml).

    Techniques: