Structured Review

GenScript open reading frame orf
Open Reading Frame Orf, supplied by GenScript, used in various techniques. Bioz Stars score: 89/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/open reading frame orf/product/GenScript
Average 89 stars, based on 6 article reviews
Price from $9.99 to $1999.99
open reading frame orf - by Bioz Stars, 2020-09
89/100 stars

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Related Articles

Sequencing:

Article Title: Detection of distinct glycosylation patterns on human γ-glutamyl transpeptidase 1 using antibody-lectin sandwich array (ALSA) technology
Article Snippet: .. Microvirin purification The nucleotide sequence for the open-reading frame (ORF) of MVN (EMBL accession AM041066) was synthesized with flanking NdeI and BamHI restriction sites in the pUC57 shuttle vector GenScript (Piscataway, NJ). .. The MVN ORF was transferred into the E. coli expression construct pET-15b (Invitrogen, Grand Island, NY), using NdeI and BamHI, where it was expected to encode the full-length protein with an N-terminal hexahistadine tag.

Purification:

Article Title: Detection of distinct glycosylation patterns on human γ-glutamyl transpeptidase 1 using antibody-lectin sandwich array (ALSA) technology
Article Snippet: .. Microvirin purification The nucleotide sequence for the open-reading frame (ORF) of MVN (EMBL accession AM041066) was synthesized with flanking NdeI and BamHI restriction sites in the pUC57 shuttle vector GenScript (Piscataway, NJ). .. The MVN ORF was transferred into the E. coli expression construct pET-15b (Invitrogen, Grand Island, NY), using NdeI and BamHI, where it was expected to encode the full-length protein with an N-terminal hexahistadine tag.

Plasmid Preparation:

Article Title: Detection of distinct glycosylation patterns on human γ-glutamyl transpeptidase 1 using antibody-lectin sandwich array (ALSA) technology
Article Snippet: .. Microvirin purification The nucleotide sequence for the open-reading frame (ORF) of MVN (EMBL accession AM041066) was synthesized with flanking NdeI and BamHI restriction sites in the pUC57 shuttle vector GenScript (Piscataway, NJ). .. The MVN ORF was transferred into the E. coli expression construct pET-15b (Invitrogen, Grand Island, NY), using NdeI and BamHI, where it was expected to encode the full-length protein with an N-terminal hexahistadine tag.

Synthesized:

Article Title: Detection of distinct glycosylation patterns on human γ-glutamyl transpeptidase 1 using antibody-lectin sandwich array (ALSA) technology
Article Snippet: .. Microvirin purification The nucleotide sequence for the open-reading frame (ORF) of MVN (EMBL accession AM041066) was synthesized with flanking NdeI and BamHI restriction sites in the pUC57 shuttle vector GenScript (Piscataway, NJ). .. The MVN ORF was transferred into the E. coli expression construct pET-15b (Invitrogen, Grand Island, NY), using NdeI and BamHI, where it was expected to encode the full-length protein with an N-terminal hexahistadine tag.

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  • 92
    GenScript ones targeting gal4
    FOFO2.0 precludes formation of supernumerary NSCs unless both FLP and mFLP5 are provided. Quantification of the number of NSCs (identified by expression of Miranda) per brain lobe in third-instar larvae of the indicated genotypes (above histograms) crossed to either both hs-FLP and hs-mFLP5 or just one of them (as indicated below graph), subjected or not to heat-shock (indicated by thermometers). One brain lobe per animal was picked at random. Histograms heights represent the mean and error bars the S.D. There was no statistically significant difference between any of the conditions. Data points shown were collected from two biological replicates (in order of histograms presented: n = 13; n = 12, p = 0.7177; n = 12, p = 0.964; n = 11, p = 0.9999; n = 11, p = 0.9899; n = 11, p = 0.9995; n = 12, p = 0.9963). * At low-frequency (0.3%) tumors were observed in heat-shocked animals carrying only hs-mFLP5 and FOFO2.0-pros shmiRs <t>-GAL4</t> shmiRs ; tumors were labeled by EGFP expression and in those cases only NSCs outside the green domain were counted. 10.7554/eLife.38393.006 Quantification of NSCs in indicated conditions.
    Ones Targeting Gal4, supplied by GenScript, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ones targeting gal4/product/GenScript
    Average 92 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
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    85
    GenScript orf transfection cdkn2a shrna
    p16 controls the transcription of the CDKN1A mRNA through AUF1. (A) Cell lysates were prepared from <t>CDKN2A</t> <t>-shRNA</t> expressing cells and their control counterparts and AUF1 mouse monoclonal antibody and mouse IgG (used as control) were utilized for immunoprecipitation. Co-precipitated RNA was purified and used for quantitative RT-PCR reactions. (B) Total RNA was purified from p16 proficient (HFSN1 and EH1) and p16-deficient (U2OS and HFSN1 expressing CDKN2A -shRNA) cells, expressing either AUF1-siRNA or control-siRNA. Transcripts for the indicated genes were amplified by quantitative RT-PCR. (C) U2OS cells were transfected with EGFP reporter containing either the wild-type (WT) or the mutated (Mut) CDKN1A ARE, while U2OS containing AUF1-siRNA as well as EH1 cells were transfected with the CDKN1A -WT-ARE construct. GFP fluorescence was measured 24 hrs <t>post-transfection.</t> Error bars represent means ± S.D. *: p -value
    Orf Transfection Cdkn2a Shrna, supplied by GenScript, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/orf transfection cdkn2a shrna/product/GenScript
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    85
    GenScript candidate δ 17 desaturase orf
    Sequence comparison of selected ω-3 desaturases. The amino acid sequences of five ω-3 desaturases were compared using the AlignX module of Vector NTI. FmD15: F. monoliforme Δ-12/Δ-15 <t>desaturase.</t> PrD17: P. ramorum <t>Δ-17</t> desaturase. PsD17: P. sojae Δ-17 desaturase. PaD17: P. aphanidermatum Δ-17 desaturase. SdD17: S. diclina Δ-17 desaturase. Lighter shaded areas indicate conserved residues. Darker shaded areas indicate identical residues
    Candidate δ 17 Desaturase Orf, supplied by GenScript, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/candidate δ 17 desaturase orf/product/GenScript
    Average 85 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    84
    GenScript superfolder gfp open reading frame
    Localizaton of <t>GFP-linked</t> proteins in B. fragilis A . Diagram of the <t>superfolder</t> GFP expression cassette of plasmid pWW3452 ( 5 ). B, E . Bacteroides ovatus and B. fragilis with pWW3452 display cytosolic localization of GFP. Second row corresponds to magnified field (scale bars = 1.5 µm) of the area indicated in red in the top row (scale bars = 5 µm). C . Modifications made to pWW3452 to create pLinkerGFP (left construct) and the addition of upstream genes to create fusion proteins with linker-GFP (right panel). N represents an additional nucleotide introduced to shift reading frame relative to N-terminal in-frame extension). D . B. ovatus expressing GFP-fused BACOVA_04502 , encoding an inulinase, which is secreted in outer membrane vesicles (OMVs indicated with yellow arrows). Second and third rows correspond to magnified images (scale bars = 1.5 µm) of the areas indicated in red and blue in the top row (scale bars = 5 µm). F . B. fragilis expressing GFP-fused BSAP-1, which is abundantly secreted in OMVs (indicated with yellow arrows). Second and third rows correspond to magnified images (scale bars = 1.5 µm) of the areas indicated in red and blue in the top row (scale bars = 5 µm). G . Agar overlay assay of BSAP-1 sensitive strain B. fragilis 1284 to growth inhibition by the indicated B. fragilis 638R strains.
    Superfolder Gfp Open Reading Frame, supplied by GenScript, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    FOFO2.0 precludes formation of supernumerary NSCs unless both FLP and mFLP5 are provided. Quantification of the number of NSCs (identified by expression of Miranda) per brain lobe in third-instar larvae of the indicated genotypes (above histograms) crossed to either both hs-FLP and hs-mFLP5 or just one of them (as indicated below graph), subjected or not to heat-shock (indicated by thermometers). One brain lobe per animal was picked at random. Histograms heights represent the mean and error bars the S.D. There was no statistically significant difference between any of the conditions. Data points shown were collected from two biological replicates (in order of histograms presented: n = 13; n = 12, p = 0.7177; n = 12, p = 0.964; n = 11, p = 0.9999; n = 11, p = 0.9899; n = 11, p = 0.9995; n = 12, p = 0.9963). * At low-frequency (0.3%) tumors were observed in heat-shocked animals carrying only hs-mFLP5 and FOFO2.0-pros shmiRs -GAL4 shmiRs ; tumors were labeled by EGFP expression and in those cases only NSCs outside the green domain were counted. 10.7554/eLife.38393.006 Quantification of NSCs in indicated conditions.

    Journal: eLife

    Article Title: Spatiotemporally controlled genetic perturbation for efficient large-scale studies of cell non-autonomous effects

    doi: 10.7554/eLife.38393

    Figure Lengend Snippet: FOFO2.0 precludes formation of supernumerary NSCs unless both FLP and mFLP5 are provided. Quantification of the number of NSCs (identified by expression of Miranda) per brain lobe in third-instar larvae of the indicated genotypes (above histograms) crossed to either both hs-FLP and hs-mFLP5 or just one of them (as indicated below graph), subjected or not to heat-shock (indicated by thermometers). One brain lobe per animal was picked at random. Histograms heights represent the mean and error bars the S.D. There was no statistically significant difference between any of the conditions. Data points shown were collected from two biological replicates (in order of histograms presented: n = 13; n = 12, p = 0.7177; n = 12, p = 0.964; n = 11, p = 0.9999; n = 11, p = 0.9899; n = 11, p = 0.9995; n = 12, p = 0.9963). * At low-frequency (0.3%) tumors were observed in heat-shocked animals carrying only hs-mFLP5 and FOFO2.0-pros shmiRs -GAL4 shmiRs ; tumors were labeled by EGFP expression and in those cases only NSCs outside the green domain were counted. 10.7554/eLife.38393.006 Quantification of NSCs in indicated conditions.

    Article Snippet: Hairpin sequences targeting pros or brat along with ones targeting GAL4 , flanked by AscI on the 5’ end and AvrII on the 3’ end, were generated by gene synthesis (GenScript).

    Techniques: Expressing, Labeling

    GAL4 miRs efficiently downregulate GAL4. hs-FLP; hs-mFLP5,FOFO2.0-pros miRs -GAL4 miRs flies were crossed with ase-GAL4,UAS-NLS::RFP (which express RFP in all CNS lineages) flies. Wandering third-instar larval brain lobes of progeny are shown. Following heat-shock, EGFP and GAL4 miRs are expressed by the FOFO construct leading to RFP-negative patches in perfect overlap with EGFP-labeled clones as expected from efficient GAL4 knock-down. Images are of a representative example obtained from two biological replicates (n > 10 per condition). Scale bar: 100 μm.

    Journal: eLife

    Article Title: Spatiotemporally controlled genetic perturbation for efficient large-scale studies of cell non-autonomous effects

    doi: 10.7554/eLife.38393

    Figure Lengend Snippet: GAL4 miRs efficiently downregulate GAL4. hs-FLP; hs-mFLP5,FOFO2.0-pros miRs -GAL4 miRs flies were crossed with ase-GAL4,UAS-NLS::RFP (which express RFP in all CNS lineages) flies. Wandering third-instar larval brain lobes of progeny are shown. Following heat-shock, EGFP and GAL4 miRs are expressed by the FOFO construct leading to RFP-negative patches in perfect overlap with EGFP-labeled clones as expected from efficient GAL4 knock-down. Images are of a representative example obtained from two biological replicates (n > 10 per condition). Scale bar: 100 μm.

    Article Snippet: Hairpin sequences targeting pros or brat along with ones targeting GAL4 , flanked by AscI on the 5’ end and AvrII on the 3’ end, were generated by gene synthesis (GenScript).

    Techniques: Construct, Labeling, Clone Assay

    FOFO2.0-mediated lineage-restricted CNS tumor generation within a single stock. ( a ) Wandering third-instar CNSs of hs-induced labeled tumors obtained with eight enhancer-FLP(D) and hs-mFLP5,FOFO2.0-pros miRs -GAL4 miRs compared with non-tumor-labeled lineages (same enhancer-FLP(D)s with hs-mFLP5,FOFO2.0-CD2 miRs -GAL4 miRs ) and background (no hs) tumor incidence. In the absence of heat-shock, tumors were occasionally induced with incomplete penetrance (inset in top right; numbers indicate frequency of CNSs devoid of tumours) but these were much smaller than those intentionally induced by heat-shock. ( b ) Wandering third-instar larval CNSs from progeny of the cross between indicated genotypes. When subject to heat-shock, extensive tumors are induced throughout the CNS (labeled in green and containing supernumerary NSCs). In the absence of heat-shock, tumors (albeit much smaller) are induced. ( a–b ) All images are maximum-intensity projections of Z-series; obtained from two biological replicates (n > 10 per condition and exact number indicated for the background condition in a – third column). Scale bar: 100 μm.

    Journal: eLife

    Article Title: Spatiotemporally controlled genetic perturbation for efficient large-scale studies of cell non-autonomous effects

    doi: 10.7554/eLife.38393

    Figure Lengend Snippet: FOFO2.0-mediated lineage-restricted CNS tumor generation within a single stock. ( a ) Wandering third-instar CNSs of hs-induced labeled tumors obtained with eight enhancer-FLP(D) and hs-mFLP5,FOFO2.0-pros miRs -GAL4 miRs compared with non-tumor-labeled lineages (same enhancer-FLP(D)s with hs-mFLP5,FOFO2.0-CD2 miRs -GAL4 miRs ) and background (no hs) tumor incidence. In the absence of heat-shock, tumors were occasionally induced with incomplete penetrance (inset in top right; numbers indicate frequency of CNSs devoid of tumours) but these were much smaller than those intentionally induced by heat-shock. ( b ) Wandering third-instar larval CNSs from progeny of the cross between indicated genotypes. When subject to heat-shock, extensive tumors are induced throughout the CNS (labeled in green and containing supernumerary NSCs). In the absence of heat-shock, tumors (albeit much smaller) are induced. ( a–b ) All images are maximum-intensity projections of Z-series; obtained from two biological replicates (n > 10 per condition and exact number indicated for the background condition in a – third column). Scale bar: 100 μm.

    Article Snippet: Hairpin sequences targeting pros or brat along with ones targeting GAL4 , flanked by AscI on the 5’ end and AvrII on the 3’ end, were generated by gene synthesis (GenScript).

    Techniques: Labeling

    p16 controls the transcription of the CDKN1A mRNA through AUF1. (A) Cell lysates were prepared from CDKN2A -shRNA expressing cells and their control counterparts and AUF1 mouse monoclonal antibody and mouse IgG (used as control) were utilized for immunoprecipitation. Co-precipitated RNA was purified and used for quantitative RT-PCR reactions. (B) Total RNA was purified from p16 proficient (HFSN1 and EH1) and p16-deficient (U2OS and HFSN1 expressing CDKN2A -shRNA) cells, expressing either AUF1-siRNA or control-siRNA. Transcripts for the indicated genes were amplified by quantitative RT-PCR. (C) U2OS cells were transfected with EGFP reporter containing either the wild-type (WT) or the mutated (Mut) CDKN1A ARE, while U2OS containing AUF1-siRNA as well as EH1 cells were transfected with the CDKN1A -WT-ARE construct. GFP fluorescence was measured 24 hrs post-transfection. Error bars represent means ± S.D. *: p -value

    Journal: PLoS ONE

    Article Title: p16INK4A Positively Regulates p21WAF1 Expression by suppressing AUF1-dependent mRNA decay

    doi: 10.1371/journal.pone.0070133

    Figure Lengend Snippet: p16 controls the transcription of the CDKN1A mRNA through AUF1. (A) Cell lysates were prepared from CDKN2A -shRNA expressing cells and their control counterparts and AUF1 mouse monoclonal antibody and mouse IgG (used as control) were utilized for immunoprecipitation. Co-precipitated RNA was purified and used for quantitative RT-PCR reactions. (B) Total RNA was purified from p16 proficient (HFSN1 and EH1) and p16-deficient (U2OS and HFSN1 expressing CDKN2A -shRNA) cells, expressing either AUF1-siRNA or control-siRNA. Transcripts for the indicated genes were amplified by quantitative RT-PCR. (C) U2OS cells were transfected with EGFP reporter containing either the wild-type (WT) or the mutated (Mut) CDKN1A ARE, while U2OS containing AUF1-siRNA as well as EH1 cells were transfected with the CDKN1A -WT-ARE construct. GFP fluorescence was measured 24 hrs post-transfection. Error bars represent means ± S.D. *: p -value

    Article Snippet: siRNA, shRNA and ORF transfection CDKN2A -shRNA expressed in pRNAT-U6/Neo vector (GenScript Corporation), pSILENCER-AUF1- siRNA and control-siRNA plasmids were used to carryout transfections using the human dermal fibroblast nucleofector kit (Amaxa Biosystems) following the protocol recommended by the manufacturer.

    Techniques: shRNA, Expressing, Immunoprecipitation, Purification, Quantitative RT-PCR, Amplification, Transfection, Construct, Fluorescence

    Sequence comparison of selected ω-3 desaturases. The amino acid sequences of five ω-3 desaturases were compared using the AlignX module of Vector NTI. FmD15: F. monoliforme Δ-12/Δ-15 desaturase. PrD17: P. ramorum Δ-17 desaturase. PsD17: P. sojae Δ-17 desaturase. PaD17: P. aphanidermatum Δ-17 desaturase. SdD17: S. diclina Δ-17 desaturase. Lighter shaded areas indicate conserved residues. Darker shaded areas indicate identical residues

    Journal: Applied Microbiology and Biotechnology

    Article Title: Identification and characterization of new ?-17 fatty acid desaturases

    doi: 10.1007/s00253-012-4068-2

    Figure Lengend Snippet: Sequence comparison of selected ω-3 desaturases. The amino acid sequences of five ω-3 desaturases were compared using the AlignX module of Vector NTI. FmD15: F. monoliforme Δ-12/Δ-15 desaturase. PrD17: P. ramorum Δ-17 desaturase. PsD17: P. sojae Δ-17 desaturase. PaD17: P. aphanidermatum Δ-17 desaturase. SdD17: S. diclina Δ-17 desaturase. Lighter shaded areas indicate conserved residues. Darker shaded areas indicate identical residues

    Article Snippet: DNA fragments encoding the codon optimized versions of candidate Δ-17 desaturase ORF were synthesized de novo by Genscript (Piscataway, NJ 08854, USA).

    Techniques: Sequencing, Plasmid Preparation

    Conversion efficiency of ω-3 desaturases on different fatty acids produced in the engineered strain. Strain L38 carrying different Δ-17 plasmids was grown in the presence of a mixture of 0.5 mM each of exogenous GLA, EDA, and ARA. Cells were harvested and fatty acid content analyzed as described in “ Materials and methods ”. Conversion efficiency is calculated as \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ {1}00\% \times {C_{\text{product}}}/\left( {{C_{\text{product}}} + {C_{\text{substrate}}}} \right) $$\end{document} , where C product and C substrate are concentrations of the product and substrate of the Δ-17 desaturase

    Journal: Applied Microbiology and Biotechnology

    Article Title: Identification and characterization of new ?-17 fatty acid desaturases

    doi: 10.1007/s00253-012-4068-2

    Figure Lengend Snippet: Conversion efficiency of ω-3 desaturases on different fatty acids produced in the engineered strain. Strain L38 carrying different Δ-17 plasmids was grown in the presence of a mixture of 0.5 mM each of exogenous GLA, EDA, and ARA. Cells were harvested and fatty acid content analyzed as described in “ Materials and methods ”. Conversion efficiency is calculated as \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ {1}00\% \times {C_{\text{product}}}/\left( {{C_{\text{product}}} + {C_{\text{substrate}}}} \right) $$\end{document} , where C product and C substrate are concentrations of the product and substrate of the Δ-17 desaturase

    Article Snippet: DNA fragments encoding the codon optimized versions of candidate Δ-17 desaturase ORF were synthesized de novo by Genscript (Piscataway, NJ 08854, USA).

    Techniques: Produced, Acetylene Reduction Assay

    Schematic diagram of the fatty acid biosynthetic pathway leading to the production of EPA. ALA α-linolenic acid, EDA eicosadienoic acid, GLA γ-linolenic acid, STA stearidonic acid, ETrA eicosatrienoic acid, DGLA dihomo-γ-linolenic acid, ETA eicosatetraenoic acid, ARA arachidonic acid, EPA eicosapentaenoic acid, Δ15D Δ-15 desaturase, Δ6D Δ-6 desaturase, ELI Δ-6 elongase, Δ9E Δ-9 elongase, Δ8D Δ-8 desaturase, Δ5D Δ-5 desaturase, Δ17D Δ-17 desaturase

    Journal: Applied Microbiology and Biotechnology

    Article Title: Identification and characterization of new ?-17 fatty acid desaturases

    doi: 10.1007/s00253-012-4068-2

    Figure Lengend Snippet: Schematic diagram of the fatty acid biosynthetic pathway leading to the production of EPA. ALA α-linolenic acid, EDA eicosadienoic acid, GLA γ-linolenic acid, STA stearidonic acid, ETrA eicosatrienoic acid, DGLA dihomo-γ-linolenic acid, ETA eicosatetraenoic acid, ARA arachidonic acid, EPA eicosapentaenoic acid, Δ15D Δ-15 desaturase, Δ6D Δ-6 desaturase, ELI Δ-6 elongase, Δ9E Δ-9 elongase, Δ8D Δ-8 desaturase, Δ5D Δ-5 desaturase, Δ17D Δ-17 desaturase

    Article Snippet: DNA fragments encoding the codon optimized versions of candidate Δ-17 desaturase ORF were synthesized de novo by Genscript (Piscataway, NJ 08854, USA).

    Techniques: Acetylene Reduction Assay

    Predicted secondary structure of P. aphanidermatum Δ-17 desaturase. Initial prediction of transmembrane (TM) domain was carried out using program TMHMM. Multiple sequence alignment was generated for a number of Δ-17 desaturases and the three conserved His-rich motifs were easily identified. The topology model was adjusted to bring the three His-rich motifs to the cytoplasmic side

    Journal: Applied Microbiology and Biotechnology

    Article Title: Identification and characterization of new ?-17 fatty acid desaturases

    doi: 10.1007/s00253-012-4068-2

    Figure Lengend Snippet: Predicted secondary structure of P. aphanidermatum Δ-17 desaturase. Initial prediction of transmembrane (TM) domain was carried out using program TMHMM. Multiple sequence alignment was generated for a number of Δ-17 desaturases and the three conserved His-rich motifs were easily identified. The topology model was adjusted to bring the three His-rich motifs to the cytoplasmic side

    Article Snippet: DNA fragments encoding the codon optimized versions of candidate Δ-17 desaturase ORF were synthesized de novo by Genscript (Piscataway, NJ 08854, USA).

    Techniques: Sequencing, Generated

    Localizaton of GFP-linked proteins in B. fragilis A . Diagram of the superfolder GFP expression cassette of plasmid pWW3452 ( 5 ). B, E . Bacteroides ovatus and B. fragilis with pWW3452 display cytosolic localization of GFP. Second row corresponds to magnified field (scale bars = 1.5 µm) of the area indicated in red in the top row (scale bars = 5 µm). C . Modifications made to pWW3452 to create pLinkerGFP (left construct) and the addition of upstream genes to create fusion proteins with linker-GFP (right panel). N represents an additional nucleotide introduced to shift reading frame relative to N-terminal in-frame extension). D . B. ovatus expressing GFP-fused BACOVA_04502 , encoding an inulinase, which is secreted in outer membrane vesicles (OMVs indicated with yellow arrows). Second and third rows correspond to magnified images (scale bars = 1.5 µm) of the areas indicated in red and blue in the top row (scale bars = 5 µm). F . B. fragilis expressing GFP-fused BSAP-1, which is abundantly secreted in OMVs (indicated with yellow arrows). Second and third rows correspond to magnified images (scale bars = 1.5 µm) of the areas indicated in red and blue in the top row (scale bars = 5 µm). G . Agar overlay assay of BSAP-1 sensitive strain B. fragilis 1284 to growth inhibition by the indicated B. fragilis 638R strains.

    Journal: bioRxiv

    Article Title: Nanaerobic growth enables direct visualization of dynamic cellular processes in human gut symbionts) Shallow breathing: bacterial life at low O(

    doi: 10.1101/2020.05.22.111492

    Figure Lengend Snippet: Localizaton of GFP-linked proteins in B. fragilis A . Diagram of the superfolder GFP expression cassette of plasmid pWW3452 ( 5 ). B, E . Bacteroides ovatus and B. fragilis with pWW3452 display cytosolic localization of GFP. Second row corresponds to magnified field (scale bars = 1.5 µm) of the area indicated in red in the top row (scale bars = 5 µm). C . Modifications made to pWW3452 to create pLinkerGFP (left construct) and the addition of upstream genes to create fusion proteins with linker-GFP (right panel). N represents an additional nucleotide introduced to shift reading frame relative to N-terminal in-frame extension). D . B. ovatus expressing GFP-fused BACOVA_04502 , encoding an inulinase, which is secreted in outer membrane vesicles (OMVs indicated with yellow arrows). Second and third rows correspond to magnified images (scale bars = 1.5 µm) of the areas indicated in red and blue in the top row (scale bars = 5 µm). F . B. fragilis expressing GFP-fused BSAP-1, which is abundantly secreted in OMVs (indicated with yellow arrows). Second and third rows correspond to magnified images (scale bars = 1.5 µm) of the areas indicated in red and blue in the top row (scale bars = 5 µm). G . Agar overlay assay of BSAP-1 sensitive strain B. fragilis 1284 to growth inhibition by the indicated B. fragilis 638R strains.

    Article Snippet: Creation of linker-gfp vector (pLinkerGFP) for generation of fusion proteins A 753-bp piece of DNA was synthesized by GenScript containing the superfolder GFP open reading frame of pWW3452 ( ) including a stop codon without the downstream Flag and His-tags and including the upstream liner region GCAGCTGCAGGAGGTGGA encoding Ala-Ala-Ala-Gly-Gly-Gly used by Basler et al . ( ) for the fusion of proteins to gfp .

    Techniques: Expressing, Plasmid Preparation, Construct, Overlay Assay, Inhibition