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  • 99
    Thermo Fisher iptg
    Detection and purification of expressed OipA. (A) OipA was detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Lane M: low molecular weight standard protein size marker (kDa); lane 1: non-induced OipA; lane 2, 3, 4, and 5: induced OipA with 1 <t>mmol/L</t> <t>IPTG</t> at t = 0, 1, 2, and 3 hr, respectively. 30-kD band were detected in induced lanes. T = 3 hr was the best time for induction. (B) Purified OipA was detected using SDS-PAGE. Lane M: low molecular weight standard protein size marker (kDa); lane 1: 30-kD band was detected as purified OipA.
    Iptg, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 5013 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore iptg
    Optimization of a XNOR circuit with the sRNA pool. ( A ) The circuit diagram for the XNOR function is shown. Inside each NOR gate, the repressor name is shown. ( B ) Truth table for a two-input XNOR gate. ( C ) The genetic design for the XNOR circuit is shown. Blunt ended lines represent the repression of promoters. The plasmid map and part sequences are provided in Supplementary Figure S1 and Table S3 . ( D ) Response of an untuned XNOR circuit where empty vector is used in place of a sRNA pool plasmid. Inputs correspond to the absence or presence of 1 mM <t>IPTG</t> (left ‘0’ / ‘1’) and <t>aTC</t> (2 ng/ml; right ‘0’ / ‘1’). ( E ) Sort gates (shown in gray) used for each input combination are shown and numbered in the order in which they were applied to the population. The sorting procedure is described in Materials and Methods. ( F ) Response of the best sRNA-tuned XNOR circuit. ( G ) The sRNA-mediated knockdown of each repressor gene in the tuned circuit. ( H ) Calculated RBS strengths for the original untuned circuit (black bars) and for the reconstructed (new RBSs) tuned circuit (white bars) (Materials and Methods). ( I ) Response of the circuit with the substituted RBSs. For all data, error bars correspond to the standard deviation of three experiments performed on different days.
    Iptg, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 13169 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore isopropyl β d thiogalactoside
    Cloning of ectodomain of human copper transporter 1 (hNdCTR1) and analysis of recombinant fusion protein. ( A ) The correspondence of the cloned nucleotide sequence to the region of the human CTR1 gene. ( B ) The growth rate of the control Escherichia coli strain and strains transformed with plasmids expressing either glutathione-S-transferase (GST) or GST-hNdCTR1. Cycles: E. coli BL21(DE3); triangles: E. coli BL21(DE3)/pGEX-4T-1; squares: E. coli BL21(DE3)/pNdCTR1. ( C ) PAG-SDS electrophoresis of crude cellular extracts from E. coli BL21(DE3)/pGEX-4T-1 (lane a) and E. coli BL21(DE3)/pNdCTR1 (lane b) after incubation with isopropyl-β- d <t>-thiogalactoside</t> (IPTG) for 3.5 h. ( D ) PAG-SDS electrophoresis and ( E ) immunoblotting with antibodies to CTR1 of crude cellular extracts from E. coli BL21(DE3)/pNdCTR1 after incubation for 0, 1.5, and 3.5 h. Lanes a, b, c: total protein extract of non-induced culture; lanes d, e, f: total protein extract of IPTG induced culture. ( F , G ) PAG-SDS electrophoretic analysis of subcellular fractions from E. coli cells transformed with pNdCTR1 and pGEX-4T-1, respectively: cell crude extract (a), cell lysate after ultrasound treatment (b), soluble fraction of lysate obtained by centrifugation for 15 min at 16,000 g (c). Insoluble fraction of pellet was 3 times treated with solution containing 1% Triton X-100, 5 mM DTT and 2 M urea. After each treatment, soluble and insoluble fractions were separated by centrifugation for 15 min at 16,000 g . Consecutive pellets: d, f, h, and corresponding to them soluble fractions: e, g. arrows show molecular weights.
    Isopropyl β D Thiogalactoside, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1315 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    iptg  (TaKaRa)
    99
    TaKaRa iptg
    3F3A mAb, but not anti-Gp78C, selectively recognizes dephosphorylated <t>Gp78.</t> (A) Cos7 cells were untransfected (Cos7 NT) or transfected with empty vector (PC3 EV), FLAG-tagged wild type (WT), S538A, phosphomimetic S538D or S538E, D536A mutant, or amino acid 533–541 deletion mutant (Δ3F3A) of Gp78. Cell lysates were immunoprecipitated with FLAG beads and probed with 3F3A, Gp78C, or FLAG mAbs. (B) Bacteria transformed with GST-tagged empty vector (GST-EV) or Gp78 C-terminal (GST-Gp78C) wild type (WT), S538A, S538D, S538E mutants, or amino acid 533–541 deletion mutant (Δ3F3A) were uninduced or induced by <t>IPTG.</t> Total bacterial lysates were dot blotted onto membranes and probed with 3F3A, Gp78C, or GST mAbs. (C) FLAG-tagged wild type of Gp78 (FLAG-Gp78-WT) or phosphorylation dominant-negative mutant (FLAG-Gp78 S538A) from Cos7 cells were immunoprecipitated and treated or not with CIAP and probed with 3F3A mAb, Gp78C, or FLAG mAb (±SEM; n = 3).
    Iptg, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 756 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher isopropyl β d thiogalactopyranoside iptg
    3F3A mAb, but not anti-Gp78C, selectively recognizes dephosphorylated <t>Gp78.</t> (A) Cos7 cells were untransfected (Cos7 NT) or transfected with empty vector (PC3 EV), FLAG-tagged wild type (WT), S538A, phosphomimetic S538D or S538E, D536A mutant, or amino acid 533–541 deletion mutant (Δ3F3A) of Gp78. Cell lysates were immunoprecipitated with FLAG beads and probed with 3F3A, Gp78C, or FLAG mAbs. (B) Bacteria transformed with GST-tagged empty vector (GST-EV) or Gp78 C-terminal (GST-Gp78C) wild type (WT), S538A, S538D, S538E mutants, or amino acid 533–541 deletion mutant (Δ3F3A) were uninduced or induced by <t>IPTG.</t> Total bacterial lysates were dot blotted onto membranes and probed with 3F3A, Gp78C, or GST mAbs. (C) FLAG-tagged wild type of Gp78 (FLAG-Gp78-WT) or phosphorylation dominant-negative mutant (FLAG-Gp78 S538A) from Cos7 cells were immunoprecipitated and treated or not with CIAP and probed with 3F3A mAb, Gp78C, or FLAG mAb (±SEM; n = 3).
    Isopropyl β D Thiogalactopyranoside Iptg, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 660 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    Thermo Fisher iptg solution ready to use
    <t>NNAT</t> expression is associated with attenuated decay of [Ca 2+ ] i following ATP-induced release of calcium stores in human osteosarcoma cells. ( A ) MNNG/HOS cells were engineered for stable, <t>IPTG-inducible</t> expression of NNATα or NNATβ as described in Methods. NNATα (HNNA) and NNATβ (HNNB) expression was assayed by immunoblot in whole cell lysates of cells with and without IPTG treatment as indicated. VC represents empty vector control non-expressors. A lysate of MNNG/HOS cells transiently transfected to express NNATα was used as a positive control. ( B ) [Ca 2+ ] i was measured by fluorescence imaging in fura-2/AM-loaded cells following ATP stimulation. HNNA cells were imaged with (blue tracing) and without IPTG (red tracing) induction of NNATα expression. VC cells are empty vector-control non-expressors imaged after IPTG treatment (green tracing). Shown are representative tracings from 3 replicate experiments.
    Iptg Solution Ready To Use, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Promega iptg
    Expression and purification of recombinant Cathepsin L-like protein. Protein samples were separated by 12.5% SDS-PAGE gel electrophoresis. ( A ) Molecular weight standard, ( B ) lysate of culture before and ( C ) after induction with <t>IPTG</t> and ( D ) recombinant Cathepsin L-like protein (MW 47.6 KDa) purified by gel filtration.
    Iptg, supplied by Promega, used in various techniques. Bioz Stars score: 93/100, based on 824 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Gold Biotechnology Inc iptg
    Evolutionary stability of a circuit encoded on a genome versus a plasmid The wiring diagrams and designs are shown. The sequences of genetic parts are provided in Appendix Table S5 . The total RNAP flux for the genome‐encoded circuit (gray) is compared to the circuit designed for a p15a plasmid (black) ( Materials and Methods ). The inducers for the genome‐encoded circuit and plasmid‐encoded circuit are 200 μM vanillic acid, 10 μM OHC14/20 ng/μl <t>aTc</t> and 2 ng/μl aTc, 0.2 mM <t>IPTG</t> and 0.1 pg/μl OHC6, respectively. Cell density measurements for cultures grown in the different circuit states (combinations of inducers). The means of three experiments performed on different days are shown, and the error bars are the standard deviation of these measurements. The cell densities are normalized by the OD 600 measured using cells without genetic circuits ( Materials and Methods ). The activities of the circuit output promoters are shown as a function of time. The data are normalized by the fluorescence of the first time point (time = 0). The shading indicates the periods where cells are grown in the presence of the combinations of inducers shown at the top. The inducers for the genome‐encoded circuit (triangles) and plasmid‐encoded circuit (circles) are 200 μM vanillic acid, 10 μM OHC14, 20 ng/μl aTc and 2 ng/μl aTc, 0.2 mM IPTG and 0.1 pg/μl OHC6, respectively. The dashed line represents the autofluorescence of wild‐type E. coli MG1655. The circuit designs and their responses to different combinations of inducers are shown in Appendix Fig S11 . The autofluorescence was measured as 0.05 (a.u.).
    Iptg, supplied by Gold Biotechnology Inc, used in various techniques. Bioz Stars score: 92/100, based on 388 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher isopropyl β d 1 thiogalactopyranoside iptg
    Evolutionary stability of a circuit encoded on a genome versus a plasmid The wiring diagrams and designs are shown. The sequences of genetic parts are provided in Appendix Table S5 . The total RNAP flux for the genome‐encoded circuit (gray) is compared to the circuit designed for a p15a plasmid (black) ( Materials and Methods ). The inducers for the genome‐encoded circuit and plasmid‐encoded circuit are 200 μM vanillic acid, 10 μM OHC14/20 ng/μl <t>aTc</t> and 2 ng/μl aTc, 0.2 mM <t>IPTG</t> and 0.1 pg/μl OHC6, respectively. Cell density measurements for cultures grown in the different circuit states (combinations of inducers). The means of three experiments performed on different days are shown, and the error bars are the standard deviation of these measurements. The cell densities are normalized by the OD 600 measured using cells without genetic circuits ( Materials and Methods ). The activities of the circuit output promoters are shown as a function of time. The data are normalized by the fluorescence of the first time point (time = 0). The shading indicates the periods where cells are grown in the presence of the combinations of inducers shown at the top. The inducers for the genome‐encoded circuit (triangles) and plasmid‐encoded circuit (circles) are 200 μM vanillic acid, 10 μM OHC14, 20 ng/μl aTc and 2 ng/μl aTc, 0.2 mM IPTG and 0.1 pg/μl OHC6, respectively. The dashed line represents the autofluorescence of wild‐type E. coli MG1655. The circuit designs and their responses to different combinations of inducers are shown in Appendix Fig S11 . The autofluorescence was measured as 0.05 (a.u.).
    Isopropyl β D 1 Thiogalactopyranoside Iptg, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 444 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Fisher Scientific iptg
    SOD activities from S. aureus sod mutants. Lane 1, RN6390; lane 2, <t>sodM</t> mutant; lane 3, sodA mutant; lane 4, double ( sodM sodA ) mutant; lane 5, double mutant containing pCL15 sodM induced with <t>IPTG;</t> lane 6, double mutant containing pCL15 sodM uninduced.
    Iptg, supplied by Fisher Scientific, used in various techniques. Bioz Stars score: 92/100, based on 430 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Carl Roth GmbH iptg
    SOD activities from S. aureus sod mutants. Lane 1, RN6390; lane 2, <t>sodM</t> mutant; lane 3, sodA mutant; lane 4, double ( sodM sodA ) mutant; lane 5, double mutant containing pCL15 sodM induced with <t>IPTG;</t> lane 6, double mutant containing pCL15 sodM uninduced.
    Iptg, supplied by Carl Roth GmbH, used in various techniques. Bioz Stars score: 92/100, based on 286 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Gold Biotechnology Inc isopropyl β d 1 thiogalactopyranoside iptg
    Construction, expression, and purification of cell-penetrating Mx1-9R. Escherichia coli cells were transformed with pET28a vector containing Mx1-9R fusion DNA sequence. After isopropyl <t>β-D-1-thiogalactopyranoside</t> <t>(IPTG)</t> induction, expressed Mx1-9R were purified by Ni-NTA column. ( A ) Structure of the Mx1-9R fusion protein was designed as described in the Methods. ( B ) Agarose gel electrophoresis of amplified Mx1-9R insert. ( C ) SDS-PAGE (2%) was performed to confirm the induction of Mx1-9R. BL21 (DE3). Lane 1: Whole lysates of control cells cultured without IPTG; lane 2: Whole cells induced with IPTG; lane 3: Purified Mx1-9R using Ni-NTA column.
    Isopropyl β D 1 Thiogalactopyranoside Iptg, supplied by Gold Biotechnology Inc, used in various techniques. Bioz Stars score: 92/100, based on 83 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Fisher Scientific isopropyl β d thiogalactopyranoside iptg
    Construction, expression, and purification of cell-penetrating Mx1-9R. Escherichia coli cells were transformed with pET28a vector containing Mx1-9R fusion DNA sequence. After isopropyl <t>β-D-1-thiogalactopyranoside</t> <t>(IPTG)</t> induction, expressed Mx1-9R were purified by Ni-NTA column. ( A ) Structure of the Mx1-9R fusion protein was designed as described in the Methods. ( B ) Agarose gel electrophoresis of amplified Mx1-9R insert. ( C ) SDS-PAGE (2%) was performed to confirm the induction of Mx1-9R. BL21 (DE3). Lane 1: Whole lysates of control cells cultured without IPTG; lane 2: Whole cells induced with IPTG; lane 3: Purified Mx1-9R using Ni-NTA column.
    Isopropyl β D Thiogalactopyranoside Iptg, supplied by Fisher Scientific, used in various techniques. Bioz Stars score: 92/100, based on 98 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Research Products International iptg
    A predictive framework for phenotypic and energetic dissection of the simple repression motif. (A) The inducible simple repression architecture. When in the active state, the repressor (gray) binds the cognate operator sequence of the DNA (red box) with high specificity, preventing transcription by occluding binding of the RNA polymerase to the promoter (blue rectangle). Upon addition of an inducer molecule, the inactive state becomes energetically preferable and the repressor no longer binds the operator sequence with appreciable specificity. Once unbound from the operator, binding of the RNA polymerase (blue) is no longer blocked and transcription can occur. (B) The simple repression input-output function for an allosteric repressor with two inducer binding sites. The key parameters are identified in speech bubbles. (C) Fold-change in gene expression collapses as a function of the free energy. The input-output function in (B) can be re-written as a Fermi function with an energetic parameter F which is the energetic difference between the repressor bound and unbound states of the promoter. Top panel shows induction profiles reported in Razo-Mejia et al. 2018 ( 10 ) of eighteen different strains over twelve concentrations of the gratuitous inducer Isopropyl <t>β</t> -D-1-thiogalactopyranoside <t>(IPTG).</t> Upon calculation of the free energy, the data collapse onto a single master curve (bottom panel) defined by F .
    Iptg, supplied by Research Products International, used in various techniques. Bioz Stars score: 93/100, based on 243 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Fisher Scientific isopropyl β d 1 thiogalactopyranoside iptg
    A predictive framework for phenotypic and energetic dissection of the simple repression motif. (A) The inducible simple repression architecture. When in the active state, the repressor (gray) binds the cognate operator sequence of the DNA (red box) with high specificity, preventing transcription by occluding binding of the RNA polymerase to the promoter (blue rectangle). Upon addition of an inducer molecule, the inactive state becomes energetically preferable and the repressor no longer binds the operator sequence with appreciable specificity. Once unbound from the operator, binding of the RNA polymerase (blue) is no longer blocked and transcription can occur. (B) The simple repression input-output function for an allosteric repressor with two inducer binding sites. The key parameters are identified in speech bubbles. (C) Fold-change in gene expression collapses as a function of the free energy. The input-output function in (B) can be re-written as a Fermi function with an energetic parameter F which is the energetic difference between the repressor bound and unbound states of the promoter. Top panel shows induction profiles reported in Razo-Mejia et al. 2018 ( 10 ) of eighteen different strains over twelve concentrations of the gratuitous inducer Isopropyl <t>β</t> -D-1-thiogalactopyranoside <t>(IPTG).</t> Upon calculation of the free energy, the data collapse onto a single master curve (bottom panel) defined by F .
    Isopropyl β D 1 Thiogalactopyranoside Iptg, supplied by Fisher Scientific, used in various techniques. Bioz Stars score: 92/100, based on 102 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Detection and purification of expressed OipA. (A) OipA was detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Lane M: low molecular weight standard protein size marker (kDa); lane 1: non-induced OipA; lane 2, 3, 4, and 5: induced OipA with 1 mmol/L IPTG at t = 0, 1, 2, and 3 hr, respectively. 30-kD band were detected in induced lanes. T = 3 hr was the best time for induction. (B) Purified OipA was detected using SDS-PAGE. Lane M: low molecular weight standard protein size marker (kDa); lane 1: 30-kD band was detected as purified OipA.

    Journal: Clinical and Experimental Vaccine Research

    Article Title: Production of specific IgY Helicobacter pylori recombinant OipA protein and assessment of its inhibitory effects towards attachment of H. pylori to AGS cell line

    doi: 10.7774/cevr.2015.4.2.177

    Figure Lengend Snippet: Detection and purification of expressed OipA. (A) OipA was detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Lane M: low molecular weight standard protein size marker (kDa); lane 1: non-induced OipA; lane 2, 3, 4, and 5: induced OipA with 1 mmol/L IPTG at t = 0, 1, 2, and 3 hr, respectively. 30-kD band were detected in induced lanes. T = 3 hr was the best time for induction. (B) Purified OipA was detected using SDS-PAGE. Lane M: low molecular weight standard protein size marker (kDa); lane 1: 30-kD band was detected as purified OipA.

    Article Snippet: Expression of recombinant protein was induced with 1 mmol/L of IPTG (Thermo Scientific, Waltham, MA, USA).

    Techniques: Purification, Polyacrylamide Gel Electrophoresis, SDS Page, Molecular Weight, Marker

    Optimization of a XNOR circuit with the sRNA pool. ( A ) The circuit diagram for the XNOR function is shown. Inside each NOR gate, the repressor name is shown. ( B ) Truth table for a two-input XNOR gate. ( C ) The genetic design for the XNOR circuit is shown. Blunt ended lines represent the repression of promoters. The plasmid map and part sequences are provided in Supplementary Figure S1 and Table S3 . ( D ) Response of an untuned XNOR circuit where empty vector is used in place of a sRNA pool plasmid. Inputs correspond to the absence or presence of 1 mM IPTG (left ‘0’ / ‘1’) and aTC (2 ng/ml; right ‘0’ / ‘1’). ( E ) Sort gates (shown in gray) used for each input combination are shown and numbered in the order in which they were applied to the population. The sorting procedure is described in Materials and Methods. ( F ) Response of the best sRNA-tuned XNOR circuit. ( G ) The sRNA-mediated knockdown of each repressor gene in the tuned circuit. ( H ) Calculated RBS strengths for the original untuned circuit (black bars) and for the reconstructed (new RBSs) tuned circuit (white bars) (Materials and Methods). ( I ) Response of the circuit with the substituted RBSs. For all data, error bars correspond to the standard deviation of three experiments performed on different days.

    Journal: Nucleic Acids Research

    Article Title: Balancing gene expression without library construction via a reusable sRNA pool

    doi: 10.1093/nar/gkx530

    Figure Lengend Snippet: Optimization of a XNOR circuit with the sRNA pool. ( A ) The circuit diagram for the XNOR function is shown. Inside each NOR gate, the repressor name is shown. ( B ) Truth table for a two-input XNOR gate. ( C ) The genetic design for the XNOR circuit is shown. Blunt ended lines represent the repression of promoters. The plasmid map and part sequences are provided in Supplementary Figure S1 and Table S3 . ( D ) Response of an untuned XNOR circuit where empty vector is used in place of a sRNA pool plasmid. Inputs correspond to the absence or presence of 1 mM IPTG (left ‘0’ / ‘1’) and aTC (2 ng/ml; right ‘0’ / ‘1’). ( E ) Sort gates (shown in gray) used for each input combination are shown and numbered in the order in which they were applied to the population. The sorting procedure is described in Materials and Methods. ( F ) Response of the best sRNA-tuned XNOR circuit. ( G ) The sRNA-mediated knockdown of each repressor gene in the tuned circuit. ( H ) Calculated RBS strengths for the original untuned circuit (black bars) and for the reconstructed (new RBSs) tuned circuit (white bars) (Materials and Methods). ( I ) Response of the circuit with the substituted RBSs. For all data, error bars correspond to the standard deviation of three experiments performed on different days.

    Article Snippet: IPTG (Sigma-Aldrich, I6758) and aTC (37919) were used as inducers.

    Techniques: Plasmid Preparation, Standard Deviation

    Analysis and characterization of expressed proteins. Notes: ( A ) SDS-PAGE gel analysis of small-scale expression of the nine designed analogs: the arrow on the right marks the position of the expected protein band; M: protein marker; A: IPTG-induced; B: uninduced. ( B ) Representative chromatographs of analog-6 (left) and PEG-analog-6 (right) eluted from the SP HP column. ( C ) SDS-PAGE gel analysis of purified proteins (left) and purified PEGylated proteins (right): the samples were undenatured and run in nonreducing conditions. ( D ) HPLC analysis of purified IFN-λ1 (IL-29S) (left) and analog-6 (right): the peak on the left represents the PEGylated protein, and the peak on the right represents the unmodified protein. Abbreviations: IFN, interferon; IPTG, isopropyl-β-D-thiogalactopyranoside; PEG-IFN, pegylated-interferon; SDS-PAGE, polyacrylamide gel electrophoresis.

    Journal: Drug Design, Development and Therapy

    Article Title: Design and evaluation of novel interferon lambda analogs with enhanced antiviral activity and improved drug attributes

    doi: 10.2147/DDDT.S91455

    Figure Lengend Snippet: Analysis and characterization of expressed proteins. Notes: ( A ) SDS-PAGE gel analysis of small-scale expression of the nine designed analogs: the arrow on the right marks the position of the expected protein band; M: protein marker; A: IPTG-induced; B: uninduced. ( B ) Representative chromatographs of analog-6 (left) and PEG-analog-6 (right) eluted from the SP HP column. ( C ) SDS-PAGE gel analysis of purified proteins (left) and purified PEGylated proteins (right): the samples were undenatured and run in nonreducing conditions. ( D ) HPLC analysis of purified IFN-λ1 (IL-29S) (left) and analog-6 (right): the peak on the left represents the PEGylated protein, and the peak on the right represents the unmodified protein. Abbreviations: IFN, interferon; IPTG, isopropyl-β-D-thiogalactopyranoside; PEG-IFN, pegylated-interferon; SDS-PAGE, polyacrylamide gel electrophoresis.

    Article Snippet: An amount of 1 mL of the culture was transferred to 4°C and the remaining 2 mL was cultured in the presence of 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) (Sigma, St Louis, MO, USA) for another 3 hours.

    Techniques: SDS Page, Expressing, Marker, Purification, High Performance Liquid Chromatography, Polyacrylamide Gel Electrophoresis

    A Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) Analysis Performed to Detect Zinc Finger Nuclease (ZFN) Expression Lanes 1 – 3, The Total Cell Protein (Tcp) of Escherichia coli top10 containing pZFN induced with 1 Mm Isopropyl Β-D-1-thiogalactopyranoside 4, 2, And 1 hour After Induction, Respectively; lane 4, the non-induced E. coli TOP10 containing pZFN; lane 5: the prestained protein ladder (Thermo Scientific, Waltham, MA, USA); lane 6, the TCP of the E. coli TOP10 containing pP15a, kanamycin-resistant ; lane 7 the TCP of E. coli TOP10 as negative controls. The ZFN protein band was not visible on SDS-PAGE due to the low levels of expression; B, thermo scientific prestained protein ladder size.

    Journal: Jundishapur Journal of Microbiology

    Article Title: Zinc Finger Nuclease: A New Approach to Overcome Beta-Lactam Antibiotic Resistance

    doi: 10.5812/jjm.29384

    Figure Lengend Snippet: A Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) Analysis Performed to Detect Zinc Finger Nuclease (ZFN) Expression Lanes 1 – 3, The Total Cell Protein (Tcp) of Escherichia coli top10 containing pZFN induced with 1 Mm Isopropyl Β-D-1-thiogalactopyranoside 4, 2, And 1 hour After Induction, Respectively; lane 4, the non-induced E. coli TOP10 containing pZFN; lane 5: the prestained protein ladder (Thermo Scientific, Waltham, MA, USA); lane 6, the TCP of the E. coli TOP10 containing pP15a, kanamycin-resistant ; lane 7 the TCP of E. coli TOP10 as negative controls. The ZFN protein band was not visible on SDS-PAGE due to the low levels of expression; B, thermo scientific prestained protein ladder size.

    Article Snippet: All media were supplemented with a final concentration of 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) (Sigma-Aldrich, MI, USA) to induce protein expression from the Lacuv5 promoters and 100 µM ZnCl2 (Merck, NJ, USA).

    Techniques: Polyacrylamide Gel Electrophoresis, SDS Page, Zinc-Fingers, Expressing

    Characterization of Cap-AuNPs. Notes: ( A ) SDS-PAGE (left) and western blot (right) analysis of Cap protein expressed in E. coli BL21 (DE3). SDS-PAGE gel was stained with silver nitrate. Lane M: molecular weight markers; Lane 1: extract of BL21 cells transformed with pET28a-Cap plasmid induced by IPTG; Lane 2: purified Cap protein. ( B ) UV-vis spectrum of AuNPs (λ max =522 nm) and Cap-AuNPs (λ max =529 nm). ( C ) Transmission electron micrograph of AuNPs. Scale bars =50 nm. ( D ) Transmission electron micrograph of Cap-AuNPs. Scale bars =50 nm. ( E ) DLS data showing size distribution of AuNPs, Cap-AuNPs, and lyophilized Cap-AuNPs. ( F ) Zeta potential of AuNPs, Cap-AuNPs, and lyophilized Cap-AuNPs (n=3 per group). ( G ) FTIR spectra of Cap and Cap-AuNPs. The S-H bonding in Cap is identified by a circle with a weak peak near 2,550 cm −1 region. Abbreviations: Cap, capsid; AuNPs, gold nanoparticles; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; E. coli , Escherichia coli ; IPTG, isopropyl-β-d-thiogalactoside; UV-vis, ultraviolet-visible spectroscopy; DLS, dynamic light scattering; FTIR, Fourier transform infrared spectroscopy.

    Journal: International Journal of Nanomedicine

    Article Title: Nanoparticle orientationally displayed antigen epitopes improve neutralizing antibody level in a model of porcine circovirus type 2

    doi: 10.2147/IJN.S140789

    Figure Lengend Snippet: Characterization of Cap-AuNPs. Notes: ( A ) SDS-PAGE (left) and western blot (right) analysis of Cap protein expressed in E. coli BL21 (DE3). SDS-PAGE gel was stained with silver nitrate. Lane M: molecular weight markers; Lane 1: extract of BL21 cells transformed with pET28a-Cap plasmid induced by IPTG; Lane 2: purified Cap protein. ( B ) UV-vis spectrum of AuNPs (λ max =522 nm) and Cap-AuNPs (λ max =529 nm). ( C ) Transmission electron micrograph of AuNPs. Scale bars =50 nm. ( D ) Transmission electron micrograph of Cap-AuNPs. Scale bars =50 nm. ( E ) DLS data showing size distribution of AuNPs, Cap-AuNPs, and lyophilized Cap-AuNPs. ( F ) Zeta potential of AuNPs, Cap-AuNPs, and lyophilized Cap-AuNPs (n=3 per group). ( G ) FTIR spectra of Cap and Cap-AuNPs. The S-H bonding in Cap is identified by a circle with a weak peak near 2,550 cm −1 region. Abbreviations: Cap, capsid; AuNPs, gold nanoparticles; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; E. coli , Escherichia coli ; IPTG, isopropyl-β-d-thiogalactoside; UV-vis, ultraviolet-visible spectroscopy; DLS, dynamic light scattering; FTIR, Fourier transform infrared spectroscopy.

    Article Snippet: Then, isopropyl-β-d-thiogalactoside (IPTG) was added to a final concentration of 0.1 mM to induce expression at 30°C for 10 h. The purification of Cap was performed by using a Ni-NTA His⋅Bind Resin (Novagen, Madison, WI, USA).

    Techniques: SDS Page, Western Blot, Staining, Molecular Weight, Transformation Assay, Plasmid Preparation, Purification, Transmission Assay, Polyacrylamide Gel Electrophoresis, Spectroscopy

    SDS-Page and Western blot analysis. Notes: ( A ) The expression analyses of E31C and E89C ngEPO analogs, using 12% SDS-PAGE. Lane 1, SM0431 protein marker (Fermentas, Vilinius, Lithuania); lanes 2 and 3, samples from Escherichia coli after and before induction with IPTG for E31C analog; and lanes 4 and 5, samples from E. coli after and before induction with IPTG for E89C analog. ( B ) The Western blot analyses of expressed ngEPO analogs. Lane 1, SM0431 protein marker (Fermentas); lane 2, E. coli transformed with pET-26b vector; lane 3, E. coli transformed with E31C cDNA before induction with IPTG; lane 4, E. coli transformed with E31C cDNA after induction with IPTG; and lane 5, E. coli transformed with E89C cDNA after induction with IPTG. Abbreviations: MW, molecular weight; ngEPO, nonglycosylated erythropoietin; SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; IPTG, isopropyl β-D-1-thiogalactopyranoside.

    Journal: Drug Design, Development and Therapy

    Article Title: Computational and nonglycosylated systems: a simpler approach for development of nanosized PEGylated proteins

    doi: 10.2147/DDDT.S98323

    Figure Lengend Snippet: SDS-Page and Western blot analysis. Notes: ( A ) The expression analyses of E31C and E89C ngEPO analogs, using 12% SDS-PAGE. Lane 1, SM0431 protein marker (Fermentas, Vilinius, Lithuania); lanes 2 and 3, samples from Escherichia coli after and before induction with IPTG for E31C analog; and lanes 4 and 5, samples from E. coli after and before induction with IPTG for E89C analog. ( B ) The Western blot analyses of expressed ngEPO analogs. Lane 1, SM0431 protein marker (Fermentas); lane 2, E. coli transformed with pET-26b vector; lane 3, E. coli transformed with E31C cDNA before induction with IPTG; lane 4, E. coli transformed with E31C cDNA after induction with IPTG; and lane 5, E. coli transformed with E89C cDNA after induction with IPTG. Abbreviations: MW, molecular weight; ngEPO, nonglycosylated erythropoietin; SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; IPTG, isopropyl β-D-1-thiogalactopyranoside.

    Article Snippet: The expression of analogs was induced using 1 mM isopropyl β-d -1-thiogalactopyranoside (IPTG) (Sigma-Aldrich, Munich, Germany) and analyzed using 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) using Mini-PROTEIN® Tetra Cell system (Bio-Rad Laboratories, Hercules, CA, USA); the gel was developed using the Coomassie blue staining technique.

    Techniques: SDS Page, Western Blot, Expressing, Marker, Transformation Assay, Positron Emission Tomography, Plasmid Preparation, Molecular Weight, Polyacrylamide Gel Electrophoresis

    Expression dynamics of SGIV VP19. (A) SDS-PAGE analysis of purified recombinant SGIV VP19. Lines M, 1, 2, 3, 4 and 5 showed protein markers, pET-VP19 (uninduced), pET-VP19 (IPTG induced), supernatant, pellet of induced pET-VP19 and the purified pET-VP19, respectively. (B) The specificity of anti-VP19 serum. SGIV or mock infected cells at 48 h p.i. were collected and centrifuged for SDS-PAGE and western blotting. Line M, 1 and 2 showed protein markers, mock and SGIV infected cells at 48 h p.i., respectively. (C) The expression pattern of VP19 during SGIV infection. Actin was chosen as the internal control. (D) VP19 was identified as a late protein. Cells were infected with SGIV at MOI of 0.5 for 6 h and 24 h under the treatment with CHX or AraC, respectively. Then cells were collected for western blotting analysis. The late protein MCP was chosen as a positive control.

    Journal: Virology Journal

    Article Title: Characterization of an envelope gene VP19 from Singapore grouper iridovirus

    doi: 10.1186/1743-422X-10-354

    Figure Lengend Snippet: Expression dynamics of SGIV VP19. (A) SDS-PAGE analysis of purified recombinant SGIV VP19. Lines M, 1, 2, 3, 4 and 5 showed protein markers, pET-VP19 (uninduced), pET-VP19 (IPTG induced), supernatant, pellet of induced pET-VP19 and the purified pET-VP19, respectively. (B) The specificity of anti-VP19 serum. SGIV or mock infected cells at 48 h p.i. were collected and centrifuged for SDS-PAGE and western blotting. Line M, 1 and 2 showed protein markers, mock and SGIV infected cells at 48 h p.i., respectively. (C) The expression pattern of VP19 during SGIV infection. Actin was chosen as the internal control. (D) VP19 was identified as a late protein. Cells were infected with SGIV at MOI of 0.5 for 6 h and 24 h under the treatment with CHX or AraC, respectively. Then cells were collected for western blotting analysis. The late protein MCP was chosen as a positive control.

    Article Snippet: After IPTG induction of E. coli BL21 (DE3) containing pET-VP19t, the recombinant fusion protein rVP19t was purified using the HisBind purification kit (Novagen, Germany) according to the manufacture’s protocol.

    Techniques: Expressing, SDS Page, Purification, Recombinant, Positron Emission Tomography, Infection, Western Blot, Positive Control

    Cloning of ectodomain of human copper transporter 1 (hNdCTR1) and analysis of recombinant fusion protein. ( A ) The correspondence of the cloned nucleotide sequence to the region of the human CTR1 gene. ( B ) The growth rate of the control Escherichia coli strain and strains transformed with plasmids expressing either glutathione-S-transferase (GST) or GST-hNdCTR1. Cycles: E. coli BL21(DE3); triangles: E. coli BL21(DE3)/pGEX-4T-1; squares: E. coli BL21(DE3)/pNdCTR1. ( C ) PAG-SDS electrophoresis of crude cellular extracts from E. coli BL21(DE3)/pGEX-4T-1 (lane a) and E. coli BL21(DE3)/pNdCTR1 (lane b) after incubation with isopropyl-β- d -thiogalactoside (IPTG) for 3.5 h. ( D ) PAG-SDS electrophoresis and ( E ) immunoblotting with antibodies to CTR1 of crude cellular extracts from E. coli BL21(DE3)/pNdCTR1 after incubation for 0, 1.5, and 3.5 h. Lanes a, b, c: total protein extract of non-induced culture; lanes d, e, f: total protein extract of IPTG induced culture. ( F , G ) PAG-SDS electrophoretic analysis of subcellular fractions from E. coli cells transformed with pNdCTR1 and pGEX-4T-1, respectively: cell crude extract (a), cell lysate after ultrasound treatment (b), soluble fraction of lysate obtained by centrifugation for 15 min at 16,000 g (c). Insoluble fraction of pellet was 3 times treated with solution containing 1% Triton X-100, 5 mM DTT and 2 M urea. After each treatment, soluble and insoluble fractions were separated by centrifugation for 15 min at 16,000 g . Consecutive pellets: d, f, h, and corresponding to them soluble fractions: e, g. arrows show molecular weights.

    Journal: Biomolecules

    Article Title: The Extracellular Domain of Human High Affinity Copper Transporter (hNdCTR1), Synthesized by E. coli Cells, Chelates Silver and Copper Ions In Vivo

    doi: 10.3390/biom7040078

    Figure Lengend Snippet: Cloning of ectodomain of human copper transporter 1 (hNdCTR1) and analysis of recombinant fusion protein. ( A ) The correspondence of the cloned nucleotide sequence to the region of the human CTR1 gene. ( B ) The growth rate of the control Escherichia coli strain and strains transformed with plasmids expressing either glutathione-S-transferase (GST) or GST-hNdCTR1. Cycles: E. coli BL21(DE3); triangles: E. coli BL21(DE3)/pGEX-4T-1; squares: E. coli BL21(DE3)/pNdCTR1. ( C ) PAG-SDS electrophoresis of crude cellular extracts from E. coli BL21(DE3)/pGEX-4T-1 (lane a) and E. coli BL21(DE3)/pNdCTR1 (lane b) after incubation with isopropyl-β- d -thiogalactoside (IPTG) for 3.5 h. ( D ) PAG-SDS electrophoresis and ( E ) immunoblotting with antibodies to CTR1 of crude cellular extracts from E. coli BL21(DE3)/pNdCTR1 after incubation for 0, 1.5, and 3.5 h. Lanes a, b, c: total protein extract of non-induced culture; lanes d, e, f: total protein extract of IPTG induced culture. ( F , G ) PAG-SDS electrophoretic analysis of subcellular fractions from E. coli cells transformed with pNdCTR1 and pGEX-4T-1, respectively: cell crude extract (a), cell lysate after ultrasound treatment (b), soluble fraction of lysate obtained by centrifugation for 15 min at 16,000 g (c). Insoluble fraction of pellet was 3 times treated with solution containing 1% Triton X-100, 5 mM DTT and 2 M urea. After each treatment, soluble and insoluble fractions were separated by centrifugation for 15 min at 16,000 g . Consecutive pellets: d, f, h, and corresponding to them soluble fractions: e, g. arrows show molecular weights.

    Article Snippet: Fusion protein synthesis was induced by isopropyl-β-d -thiogalactoside (IPTG, Sigma, St. Louis, MO, USA).

    Techniques: Clone Assay, Recombinant, Sequencing, Transformation Assay, Expressing, Electrophoresis, Incubation, Centrifugation

    3F3A mAb, but not anti-Gp78C, selectively recognizes dephosphorylated Gp78. (A) Cos7 cells were untransfected (Cos7 NT) or transfected with empty vector (PC3 EV), FLAG-tagged wild type (WT), S538A, phosphomimetic S538D or S538E, D536A mutant, or amino acid 533–541 deletion mutant (Δ3F3A) of Gp78. Cell lysates were immunoprecipitated with FLAG beads and probed with 3F3A, Gp78C, or FLAG mAbs. (B) Bacteria transformed with GST-tagged empty vector (GST-EV) or Gp78 C-terminal (GST-Gp78C) wild type (WT), S538A, S538D, S538E mutants, or amino acid 533–541 deletion mutant (Δ3F3A) were uninduced or induced by IPTG. Total bacterial lysates were dot blotted onto membranes and probed with 3F3A, Gp78C, or GST mAbs. (C) FLAG-tagged wild type of Gp78 (FLAG-Gp78-WT) or phosphorylation dominant-negative mutant (FLAG-Gp78 S538A) from Cos7 cells were immunoprecipitated and treated or not with CIAP and probed with 3F3A mAb, Gp78C, or FLAG mAb (±SEM; n = 3).

    Journal: Molecular Biology of the Cell

    Article Title: p38 MAP kinase–dependent phosphorylation of the Gp78 E3 ubiquitin ligase controls ER–mitochondria association and mitochondria motility

    doi: 10.1091/mbc.E15-02-0120

    Figure Lengend Snippet: 3F3A mAb, but not anti-Gp78C, selectively recognizes dephosphorylated Gp78. (A) Cos7 cells were untransfected (Cos7 NT) or transfected with empty vector (PC3 EV), FLAG-tagged wild type (WT), S538A, phosphomimetic S538D or S538E, D536A mutant, or amino acid 533–541 deletion mutant (Δ3F3A) of Gp78. Cell lysates were immunoprecipitated with FLAG beads and probed with 3F3A, Gp78C, or FLAG mAbs. (B) Bacteria transformed with GST-tagged empty vector (GST-EV) or Gp78 C-terminal (GST-Gp78C) wild type (WT), S538A, S538D, S538E mutants, or amino acid 533–541 deletion mutant (Δ3F3A) were uninduced or induced by IPTG. Total bacterial lysates were dot blotted onto membranes and probed with 3F3A, Gp78C, or GST mAbs. (C) FLAG-tagged wild type of Gp78 (FLAG-Gp78-WT) or phosphorylation dominant-negative mutant (FLAG-Gp78 S538A) from Cos7 cells were immunoprecipitated and treated or not with CIAP and probed with 3F3A mAb, Gp78C, or FLAG mAb (±SEM; n = 3).

    Article Snippet: GST-Gp78C protein expression was induced by 1 mM isopropyl-β-d -thiogalactoside (IPTG) at 37°C for 4 h. pE-SUMO His-Gp78 was purchased from LifeSensors and the His-hGp78C induced with 0.4 mM IPTG at 16°C overnight and purified with His60 Ni Superflow Resin (Clontech). pcDNA3-Flag dominant-negative MKK6(K82A) and constitutively active MKK6 (Glu) were gifts from Roger Davis (University of Massachusetts, Worcester, MA; Addgene plasmids 13519 and 13518; ).

    Techniques: Transfection, Plasmid Preparation, Mutagenesis, Immunoprecipitation, Transformation Assay, Dominant Negative Mutation

    NNAT expression is associated with attenuated decay of [Ca 2+ ] i following ATP-induced release of calcium stores in human osteosarcoma cells. ( A ) MNNG/HOS cells were engineered for stable, IPTG-inducible expression of NNATα or NNATβ as described in Methods. NNATα (HNNA) and NNATβ (HNNB) expression was assayed by immunoblot in whole cell lysates of cells with and without IPTG treatment as indicated. VC represents empty vector control non-expressors. A lysate of MNNG/HOS cells transiently transfected to express NNATα was used as a positive control. ( B ) [Ca 2+ ] i was measured by fluorescence imaging in fura-2/AM-loaded cells following ATP stimulation. HNNA cells were imaged with (blue tracing) and without IPTG (red tracing) induction of NNATα expression. VC cells are empty vector-control non-expressors imaged after IPTG treatment (green tracing). Shown are representative tracings from 3 replicate experiments.

    Journal: Oncotarget

    Article Title: Aberrant epigenetic silencing of neuronatin is a frequent event in human osteosarcoma

    doi: 10.18632/oncotarget.27583

    Figure Lengend Snippet: NNAT expression is associated with attenuated decay of [Ca 2+ ] i following ATP-induced release of calcium stores in human osteosarcoma cells. ( A ) MNNG/HOS cells were engineered for stable, IPTG-inducible expression of NNATα or NNATβ as described in Methods. NNATα (HNNA) and NNATβ (HNNB) expression was assayed by immunoblot in whole cell lysates of cells with and without IPTG treatment as indicated. VC represents empty vector control non-expressors. A lysate of MNNG/HOS cells transiently transfected to express NNATα was used as a positive control. ( B ) [Ca 2+ ] i was measured by fluorescence imaging in fura-2/AM-loaded cells following ATP stimulation. HNNA cells were imaged with (blue tracing) and without IPTG (red tracing) induction of NNATα expression. VC cells are empty vector-control non-expressors imaged after IPTG treatment (green tracing). Shown are representative tracings from 3 replicate experiments.

    Article Snippet: Cell lines exhibiting negligible NNAT expression at baseline with robust NNAT expression upon treatment with isopropyl β-D-1-thiogalactopyranoside (IPTG) were utilized for experiments.

    Techniques: Expressing, Plasmid Preparation, Transfection, Positive Control, Fluorescence, Imaging

    Expression and purification of recombinant Cathepsin L-like protein. Protein samples were separated by 12.5% SDS-PAGE gel electrophoresis. ( A ) Molecular weight standard, ( B ) lysate of culture before and ( C ) after induction with IPTG and ( D ) recombinant Cathepsin L-like protein (MW 47.6 KDa) purified by gel filtration.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Improving Serodiagnosis of Human and Canine Leishmaniasis with Recombinant Leishmania braziliensis Cathepsin L-like Protein and a Synthetic Peptide Containing Its Linear B-cell Epitope

    doi: 10.1371/journal.pntd.0003426

    Figure Lengend Snippet: Expression and purification of recombinant Cathepsin L-like protein. Protein samples were separated by 12.5% SDS-PAGE gel electrophoresis. ( A ) Molecular weight standard, ( B ) lysate of culture before and ( C ) after induction with IPTG and ( D ) recombinant Cathepsin L-like protein (MW 47.6 KDa) purified by gel filtration.

    Article Snippet: The recombinant CatL (r CATL) expression was performed by adding 1.0 mM IPTG (Isopropyl-β-D-thiogalactopyranoside, Promega, Canada) for 24 h at 12°C with shaking at 200 rev min−1 .

    Techniques: Expressing, Purification, Recombinant, SDS Page, Nucleic Acid Electrophoresis, Molecular Weight, Filtration

    Evolutionary stability of a circuit encoded on a genome versus a plasmid The wiring diagrams and designs are shown. The sequences of genetic parts are provided in Appendix Table S5 . The total RNAP flux for the genome‐encoded circuit (gray) is compared to the circuit designed for a p15a plasmid (black) ( Materials and Methods ). The inducers for the genome‐encoded circuit and plasmid‐encoded circuit are 200 μM vanillic acid, 10 μM OHC14/20 ng/μl aTc and 2 ng/μl aTc, 0.2 mM IPTG and 0.1 pg/μl OHC6, respectively. Cell density measurements for cultures grown in the different circuit states (combinations of inducers). The means of three experiments performed on different days are shown, and the error bars are the standard deviation of these measurements. The cell densities are normalized by the OD 600 measured using cells without genetic circuits ( Materials and Methods ). The activities of the circuit output promoters are shown as a function of time. The data are normalized by the fluorescence of the first time point (time = 0). The shading indicates the periods where cells are grown in the presence of the combinations of inducers shown at the top. The inducers for the genome‐encoded circuit (triangles) and plasmid‐encoded circuit (circles) are 200 μM vanillic acid, 10 μM OHC14, 20 ng/μl aTc and 2 ng/μl aTc, 0.2 mM IPTG and 0.1 pg/μl OHC6, respectively. The dashed line represents the autofluorescence of wild‐type E. coli MG1655. The circuit designs and their responses to different combinations of inducers are shown in Appendix Fig S11 . The autofluorescence was measured as 0.05 (a.u.).

    Journal: Molecular Systems Biology

    Article Title: Precision design of stable genetic circuits carried in highly‐insulated E. coli genomic landing pads

    doi: 10.15252/msb.20209584

    Figure Lengend Snippet: Evolutionary stability of a circuit encoded on a genome versus a plasmid The wiring diagrams and designs are shown. The sequences of genetic parts are provided in Appendix Table S5 . The total RNAP flux for the genome‐encoded circuit (gray) is compared to the circuit designed for a p15a plasmid (black) ( Materials and Methods ). The inducers for the genome‐encoded circuit and plasmid‐encoded circuit are 200 μM vanillic acid, 10 μM OHC14/20 ng/μl aTc and 2 ng/μl aTc, 0.2 mM IPTG and 0.1 pg/μl OHC6, respectively. Cell density measurements for cultures grown in the different circuit states (combinations of inducers). The means of three experiments performed on different days are shown, and the error bars are the standard deviation of these measurements. The cell densities are normalized by the OD 600 measured using cells without genetic circuits ( Materials and Methods ). The activities of the circuit output promoters are shown as a function of time. The data are normalized by the fluorescence of the first time point (time = 0). The shading indicates the periods where cells are grown in the presence of the combinations of inducers shown at the top. The inducers for the genome‐encoded circuit (triangles) and plasmid‐encoded circuit (circles) are 200 μM vanillic acid, 10 μM OHC14, 20 ng/μl aTc and 2 ng/μl aTc, 0.2 mM IPTG and 0.1 pg/μl OHC6, respectively. The dashed line represents the autofluorescence of wild‐type E. coli MG1655. The circuit designs and their responses to different combinations of inducers are shown in Appendix Fig S11 . The autofluorescence was measured as 0.05 (a.u.).

    Article Snippet: Then, either 12.5 mM L‐arabinose (Sigma‐Aldrich, USA, A3256), 1 mM IPTG (GoldBio, USA, I2481C), 20 ng/μl aTc (Sigma‐Aldrich, USA, 37919), 500 μM 4‐isopropylbenzoic acid (Sigma‐Aldrich, USA, 268402), 200 μM vanillic acid (Sigma‐Aldrich, USA, H36001), 10 μM OHC14 (Sigma‐Aldrich, USA, 51481), or 1 mM naringenin (Sigma‐Aldrich, USA, N5893) was used to induce the sensors.

    Techniques: Plasmid Preparation, Standard Deviation, Fluorescence

    SOD activities from S. aureus sod mutants. Lane 1, RN6390; lane 2, sodM mutant; lane 3, sodA mutant; lane 4, double ( sodM sodA ) mutant; lane 5, double mutant containing pCL15 sodM induced with IPTG; lane 6, double mutant containing pCL15 sodM uninduced.

    Journal: Journal of Bacteriology

    Article Title: Identification and Characterization of a Second Superoxide Dismutase Gene (sodM) from Staphylococcus aureus

    doi: 10.1128/JB.183.11.3399-3407.2001

    Figure Lengend Snippet: SOD activities from S. aureus sod mutants. Lane 1, RN6390; lane 2, sodM mutant; lane 3, sodA mutant; lane 4, double ( sodM sodA ) mutant; lane 5, double mutant containing pCL15 sodM induced with IPTG; lane 6, double mutant containing pCL15 sodM uninduced.

    Article Snippet: Expression of sodM was induced with IPTG (isopropyl-β- d -thiogalactopyranoside, 0.5 mM; Fisher Scientific, Fairlawn, N.J.) 1 h postinoculation, and cells were harvested 5 h later.

    Techniques: Mutagenesis

    Construction, expression, and purification of cell-penetrating Mx1-9R. Escherichia coli cells were transformed with pET28a vector containing Mx1-9R fusion DNA sequence. After isopropyl β-D-1-thiogalactopyranoside (IPTG) induction, expressed Mx1-9R were purified by Ni-NTA column. ( A ) Structure of the Mx1-9R fusion protein was designed as described in the Methods. ( B ) Agarose gel electrophoresis of amplified Mx1-9R insert. ( C ) SDS-PAGE (2%) was performed to confirm the induction of Mx1-9R. BL21 (DE3). Lane 1: Whole lysates of control cells cultured without IPTG; lane 2: Whole cells induced with IPTG; lane 3: Purified Mx1-9R using Ni-NTA column.

    Journal: Viruses

    Article Title: Cell-Penetrating Mx1 Enhances Anti-Viral Resistance against Mucosal Influenza Viral Infection

    doi: 10.3390/v11020109

    Figure Lengend Snippet: Construction, expression, and purification of cell-penetrating Mx1-9R. Escherichia coli cells were transformed with pET28a vector containing Mx1-9R fusion DNA sequence. After isopropyl β-D-1-thiogalactopyranoside (IPTG) induction, expressed Mx1-9R were purified by Ni-NTA column. ( A ) Structure of the Mx1-9R fusion protein was designed as described in the Methods. ( B ) Agarose gel electrophoresis of amplified Mx1-9R insert. ( C ) SDS-PAGE (2%) was performed to confirm the induction of Mx1-9R. BL21 (DE3). Lane 1: Whole lysates of control cells cultured without IPTG; lane 2: Whole cells induced with IPTG; lane 3: Purified Mx1-9R using Ni-NTA column.

    Article Snippet: Expression and Purification of Mx1-9R Fusion Protein Transformed BL21 cells were cultured in Luria-Bertani (LB) broth (MB Cell, Los Angeles, CA, USA) containing 30 μg/mL of kanamycin (LPS solution, Daejeon, Korea) for 3 h at 37 °C, then incubated with 0.1 mM of Isopropyl β-D-1-thiogalactopyranoside (IPTG) (Goldbio, St. Louis, MO, USA) at 18 °C overnight.

    Techniques: Expressing, Purification, Transformation Assay, Plasmid Preparation, Sequencing, Agarose Gel Electrophoresis, Amplification, SDS Page, Cell Culture

    A predictive framework for phenotypic and energetic dissection of the simple repression motif. (A) The inducible simple repression architecture. When in the active state, the repressor (gray) binds the cognate operator sequence of the DNA (red box) with high specificity, preventing transcription by occluding binding of the RNA polymerase to the promoter (blue rectangle). Upon addition of an inducer molecule, the inactive state becomes energetically preferable and the repressor no longer binds the operator sequence with appreciable specificity. Once unbound from the operator, binding of the RNA polymerase (blue) is no longer blocked and transcription can occur. (B) The simple repression input-output function for an allosteric repressor with two inducer binding sites. The key parameters are identified in speech bubbles. (C) Fold-change in gene expression collapses as a function of the free energy. The input-output function in (B) can be re-written as a Fermi function with an energetic parameter F which is the energetic difference between the repressor bound and unbound states of the promoter. Top panel shows induction profiles reported in Razo-Mejia et al. 2018 ( 10 ) of eighteen different strains over twelve concentrations of the gratuitous inducer Isopropyl β -D-1-thiogalactopyranoside (IPTG). Upon calculation of the free energy, the data collapse onto a single master curve (bottom panel) defined by F .

    Journal: bioRxiv

    Article Title: The Energetics of Molecular Adaptation in Transcriptional Regulation

    doi: 10.1101/638270

    Figure Lengend Snippet: A predictive framework for phenotypic and energetic dissection of the simple repression motif. (A) The inducible simple repression architecture. When in the active state, the repressor (gray) binds the cognate operator sequence of the DNA (red box) with high specificity, preventing transcription by occluding binding of the RNA polymerase to the promoter (blue rectangle). Upon addition of an inducer molecule, the inactive state becomes energetically preferable and the repressor no longer binds the operator sequence with appreciable specificity. Once unbound from the operator, binding of the RNA polymerase (blue) is no longer blocked and transcription can occur. (B) The simple repression input-output function for an allosteric repressor with two inducer binding sites. The key parameters are identified in speech bubbles. (C) Fold-change in gene expression collapses as a function of the free energy. The input-output function in (B) can be re-written as a Fermi function with an energetic parameter F which is the energetic difference between the repressor bound and unbound states of the promoter. Top panel shows induction profiles reported in Razo-Mejia et al. 2018 ( 10 ) of eighteen different strains over twelve concentrations of the gratuitous inducer Isopropyl β -D-1-thiogalactopyranoside (IPTG). Upon calculation of the free energy, the data collapse onto a single master curve (bottom panel) defined by F .

    Article Snippet: After approximately 12 to 15 hr, the cultures reached saturation and were diluted 1000-fold into a second 2 mL 96-deep-well plate where each well contained 500 µ L of M9 minimal media supplemented with 0.5% w/v glucose (anhydrous D-Glucose, Macron Chemicals) and the appropriate concentration of IPTG (Isopropyl β -D-1-thiogalactopyranoside, Dioxane Free, Research Products International).

    Techniques: Dissection, Sequencing, Binding Assay, Expressing