dpn i New England Biolabs Search Results


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  • 94
    New England Biolabs dpn
    Dpn, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 104 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore nadh
    Cell-free biotransformation of morphine at various ratios of MDH to MR in the presence and absence of STH. The transformations were performed without (A, C, and E) or with (B, D, and F) 1.25 U of STH per ml. MDH and MR were used as the ratios 1:1 (1.25 U of each per ml) (A and B), 5:1 (6.25 U of MDH per ml and 1.25 U of MR per ml (C and D), 1:5 (1.25 U of MDH per ml and 6.25 U of MR per ml (E and F). The cofactors <t>NADPH</t> and <t>NAD</t> + were supplied at a concentration of 0.2 mM.
    Nadh, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 4930 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    TaKaRa dpn i
    Cell-free biotransformation of morphine at various ratios of MDH to MR in the presence and absence of STH. The transformations were performed without (A, C, and E) or with (B, D, and F) 1.25 U of STH per ml. MDH and MR were used as the ratios 1:1 (1.25 U of each per ml) (A and B), 5:1 (6.25 U of MDH per ml and 1.25 U of MR per ml (C and D), 1:5 (1.25 U of MDH per ml and 6.25 U of MR per ml (E and F). The cofactors <t>NADPH</t> and <t>NAD</t> + were supplied at a concentration of 0.2 mM.
    Dpn I, supplied by TaKaRa, used in various techniques. Bioz Stars score: 95/100, based on 474 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    New England Biolabs s1 nuclease new england biolabs
    Cell-free biotransformation of morphine at various ratios of MDH to MR in the presence and absence of STH. The transformations were performed without (A, C, and E) or with (B, D, and F) 1.25 U of STH per ml. MDH and MR were used as the ratios 1:1 (1.25 U of each per ml) (A and B), 5:1 (6.25 U of MDH per ml and 1.25 U of MR per ml (C and D), 1:5 (1.25 U of MDH per ml and 6.25 U of MR per ml (E and F). The cofactors <t>NADPH</t> and <t>NAD</t> + were supplied at a concentration of 0.2 mM.
    S1 Nuclease New England Biolabs, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 90/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    dpn i  (Roche)
    92
    Roche dpn i
    Cell-free biotransformation of morphine at various ratios of MDH to MR in the presence and absence of STH. The transformations were performed without (A, C, and E) or with (B, D, and F) 1.25 U of STH per ml. MDH and MR were used as the ratios 1:1 (1.25 U of each per ml) (A and B), 5:1 (6.25 U of MDH per ml and 1.25 U of MR per ml (C and D), 1:5 (1.25 U of MDH per ml and 6.25 U of MR per ml (E and F). The cofactors <t>NADPH</t> and <t>NAD</t> + were supplied at a concentration of 0.2 mM.
    Dpn I, supplied by Roche, used in various techniques. Bioz Stars score: 92/100, based on 37 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Codexis nadh
    Cell-free biotransformation of morphine at various ratios of MDH to MR in the presence and absence of STH. The transformations were performed without (A, C, and E) or with (B, D, and F) 1.25 U of STH per ml. MDH and MR were used as the ratios 1:1 (1.25 U of each per ml) (A and B), 5:1 (6.25 U of MDH per ml and 1.25 U of MR per ml (C and D), 1:5 (1.25 U of MDH per ml and 6.25 U of MR per ml (E and F). The cofactors <t>NADPH</t> and <t>NAD</t> + were supplied at a concentration of 0.2 mM.
    Nadh, supplied by Codexis, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Biomol GmbH nadh
    <t>NAD</t> + -dependent DHPS oxidation by recombinant dehydrogenase DhpA. The reactions contained 0.85 μg/ml DhpA in 50 mM Tris–HCl buffer at pH 9.5. The DHPS concentration was varied in the presence of 10 mM NAD + and the initial rates of <t>NADH</t> formation were determined spectrophotometrically as increase of absorbance at 340 nm. The data shown represents the mean ± SD of three technical replicates.
    Nadh, supplied by Biomol GmbH, used in various techniques. Bioz Stars score: 92/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Agilent technologies dpn i
    <t>NAD</t> + -dependent DHPS oxidation by recombinant dehydrogenase DhpA. The reactions contained 0.85 μg/ml DhpA in 50 mM Tris–HCl buffer at pH 9.5. The DHPS concentration was varied in the presence of 10 mM NAD + and the initial rates of <t>NADH</t> formation were determined spectrophotometrically as increase of absorbance at 340 nm. The data shown represents the mean ± SD of three technical replicates.
    Dpn I, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 93/100, based on 189 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Stratagene dpn i
    <t>NAD</t> + -dependent DHPS oxidation by recombinant dehydrogenase DhpA. The reactions contained 0.85 μg/ml DhpA in 50 mM Tris–HCl buffer at pH 9.5. The DHPS concentration was varied in the presence of 10 mM NAD + and the initial rates of <t>NADH</t> formation were determined spectrophotometrically as increase of absorbance at 340 nm. The data shown represents the mean ± SD of three technical replicates.
    Dpn I, supplied by Stratagene, used in various techniques. Bioz Stars score: 92/100, based on 292 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Thermo Fisher dpn i enzyme
    <t>NAD</t> + -dependent DHPS oxidation by recombinant dehydrogenase DhpA. The reactions contained 0.85 μg/ml DhpA in 50 mM Tris–HCl buffer at pH 9.5. The DHPS concentration was varied in the presence of 10 mM NAD + and the initial rates of <t>NADH</t> formation were determined spectrophotometrically as increase of absorbance at 340 nm. The data shown represents the mean ± SD of three technical replicates.
    Dpn I Enzyme, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 59 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher fastdigest dpn i
    <t>NAD</t> + -dependent DHPS oxidation by recombinant dehydrogenase DhpA. The reactions contained 0.85 μg/ml DhpA in 50 mM Tris–HCl buffer at pH 9.5. The DHPS concentration was varied in the presence of 10 mM NAD + and the initial rates of <t>NADH</t> formation were determined spectrophotometrically as increase of absorbance at 340 nm. The data shown represents the mean ± SD of three technical replicates.
    Fastdigest Dpn I, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Stratagene dpn
    <t>NAD</t> + -dependent DHPS oxidation by recombinant dehydrogenase DhpA. The reactions contained 0.85 μg/ml DhpA in 50 mM Tris–HCl buffer at pH 9.5. The DHPS concentration was varied in the presence of 10 mM NAD + and the initial rates of <t>NADH</t> formation were determined spectrophotometrically as increase of absorbance at 340 nm. The data shown represents the mean ± SD of three technical replicates.
    Dpn, supplied by Stratagene, used in various techniques. Bioz Stars score: 91/100, based on 41 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    nadh  (Roche)
    93
    Roche nadh
    Determination of relative efficiencies of transcription initiation with <t>NAD</t> + vs. transcription initiation with ATP A. dsDNA transcription templates containing RNAI and T7A1 promoters (positions −40 to +3; promoter elements and transcription start sites in gray boxes); B. Representative raw data (initial RNA products of transcription reactions performed in the presence of 1 mM NAD + , 0.01–0.5 mM ATP, and [α 32 P]-CTP as extending nucleotide. C and D. Relative efficiencies of transcription initiation with NAD + vs. transcription initiation with ATP [(k cat /K M , NCIN)/(k cat /K M , ATP)]. Calculation using logarithmic regression (C; best-fit line for data points with NAD + pC/(pppApC + NAD + pC) values between 0.2 and 0.8 in (C) or non-linear regression (D); best-fit curve for data points with NAD + pC/(pppApC + NAD + pC) values between 0 and 1.
    Nadh, supplied by Roche, used in various techniques. Bioz Stars score: 93/100, based on 827 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Promega nad nadh glotm assay kit
    Schematic representation of the genes involved in solvent tolerance from megaplasmid pTTS12. SrpABC efflux pump is the major contributor of solvent tolerance trait from the megaplasmid pTTS12. This efflux pump is able to efficiently extrude solvents from membrane lipid bilayer. A COG5654-COG5642 family toxin-antitoxin system (SlvT and SlvA respectively) promoted the growth of P. putida S12 in the presence of low solvent concentration. In the absence of SlvA, SlvT causes toxicity by conferring cellular <t>NAD</t> + depletion.
    Nad Nadh Glotm Assay Kit, supplied by Promega, used in various techniques. Bioz Stars score: 91/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    New England Biolabs dpn 1
    Schematic representation of the genes involved in solvent tolerance from megaplasmid pTTS12. SrpABC efflux pump is the major contributor of solvent tolerance trait from the megaplasmid pTTS12. This efflux pump is able to efficiently extrude solvents from membrane lipid bilayer. A COG5654-COG5642 family toxin-antitoxin system (SlvT and SlvA respectively) promoted the growth of P. putida S12 in the presence of low solvent concentration. In the absence of SlvA, SlvT causes toxicity by conferring cellular <t>NAD</t> + depletion.
    Dpn 1, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 88/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Millipore β nad
    TNT is a novel <t>β-NAD</t> + -glycohydrolase of M. tuberculosis ( a ) NAD + -glycohydrolase assay for wt TNT in the absence and presence of TNT antibody (400 ng) or IFT (100 ng).( b ) The kinetics of NAD + hydrolysis by TNT (10 nM) was determined using a fluorometric assay at pH 7.4. The values of K m and V max were determined by non-linear regression analysis using the Michaelis-Menten equation. ( c ) NAD + -glycohydrolase activity of TNT (10 nM) after heating to 65°C or 95°C for 5 minutes. The initial velocity v 0 of NAD + hydrolysis was determined at a substrate concentration of 200 μM. All data in this figure represent the means ± s.e.m. of three independent experiments.
    β Nad, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 393 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs dpn i
    Genetic and growth characteristics displayed by dam- complemented mutant strains of UPEC relative to wild-type . (A) Dam methylation pattern in UPEC CFT073 strain subsequent to digestion with <t>Dpn</t> I (lane 1) and Mbo I (lane 2). The 1 kb plus DNA ladder (MW) is also shown. (B) Growth curve (CFU/milliliter versus time) for dam complement UPEC strains of CFT073, CFT073 Δ dam , cC119, and cC119 Δ dam . (C) Micrographs for wild-type (WT) and dam mutant (Δ dam ) UPEC strains, illustrating the morphological occurrence of shortened- and filamentous rods, respectively. (D) Semi-quantitative RT-PCR for mdh, rec A, and arc A expression at cycles 23, 25, and 30 for CFT073 (lanes 1–3), CFT073 Δ dam (lanes 4–6), CFT073 + pGEMdam (lanes 7–9), CFT073 Δ dam + pGEMdam (lanes 10–12). The 100 bp molecular marker MW (Promega, WI, USA) and negative control are shown (lane 13).
    Dpn I, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 2196 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher dpn i
    Genetic and growth characteristics displayed by dam- complemented mutant strains of UPEC relative to wild-type . (A) Dam methylation pattern in UPEC CFT073 strain subsequent to digestion with <t>Dpn</t> I (lane 1) and Mbo I (lane 2). The 1 kb plus DNA ladder (MW) is also shown. (B) Growth curve (CFU/milliliter versus time) for dam complement UPEC strains of CFT073, CFT073 Δ dam , cC119, and cC119 Δ dam . (C) Micrographs for wild-type (WT) and dam mutant (Δ dam ) UPEC strains, illustrating the morphological occurrence of shortened- and filamentous rods, respectively. (D) Semi-quantitative RT-PCR for mdh, rec A, and arc A expression at cycles 23, 25, and 30 for CFT073 (lanes 1–3), CFT073 Δ dam (lanes 4–6), CFT073 + pGEMdam (lanes 7–9), CFT073 Δ dam + pGEMdam (lanes 10–12). The 100 bp molecular marker MW (Promega, WI, USA) and negative control are shown (lane 13).
    Dpn I, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 815 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    New England Biolabs dpn i enzyme
    Chimera construction or insertion with a short DNA fragment . To replace a short stretch of DNA, such as a DNA fragment encoding a transmembrane region of a nAChR subunit for chimera construction, or to insert a short tag, such as FLAG-tag, into some part of a protein, amplification of the insert is not necessary. In this case, the insert can be directly included in two primers for single <t>PCR</t> amplification of the cDNA along with the vector. The forward primer starts immediately downstream of the insertion site and has a tail with the 3' part of the insert. The reverse primer starts immediately upstream of the insertion site and has a tail with 5' part of the insert. Two primers only require ~16-base overlap. Thus, for a 120 bp insertion, each primer needs to have a 68-base tail for insertion and ~17-22 bases for annealing (depending on the GC content). The total length of each primer will be about 85-90 bases. <t>Dpn</t> I digestion and transformation are the same as in Figure 1.
    Dpn I Enzyme, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 88/100, based on 135 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    New England Biolabs restriction enzyme dpn
    Chimera construction or insertion with a short DNA fragment . To replace a short stretch of DNA, such as a DNA fragment encoding a transmembrane region of a nAChR subunit for chimera construction, or to insert a short tag, such as FLAG-tag, into some part of a protein, amplification of the insert is not necessary. In this case, the insert can be directly included in two primers for single <t>PCR</t> amplification of the cDNA along with the vector. The forward primer starts immediately downstream of the insertion site and has a tail with the 3' part of the insert. The reverse primer starts immediately upstream of the insertion site and has a tail with 5' part of the insert. Two primers only require ~16-base overlap. Thus, for a 120 bp insertion, each primer needs to have a 68-base tail for insertion and ~17-22 bases for annealing (depending on the GC content). The total length of each primer will be about 85-90 bases. <t>Dpn</t> I digestion and transformation are the same as in Figure 1.
    Restriction Enzyme Dpn, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 88/100, based on 31 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Cell-free biotransformation of morphine at various ratios of MDH to MR in the presence and absence of STH. The transformations were performed without (A, C, and E) or with (B, D, and F) 1.25 U of STH per ml. MDH and MR were used as the ratios 1:1 (1.25 U of each per ml) (A and B), 5:1 (6.25 U of MDH per ml and 1.25 U of MR per ml (C and D), 1:5 (1.25 U of MDH per ml and 6.25 U of MR per ml (E and F). The cofactors NADPH and NAD + were supplied at a concentration of 0.2 mM.

    Journal: Applied and Environmental Microbiology

    Article Title: Cofactor Regeneration by a Soluble Pyridine Nucleotide Transhydrogenase for Biological Production of Hydromorphone

    doi:

    Figure Lengend Snippet: Cell-free biotransformation of morphine at various ratios of MDH to MR in the presence and absence of STH. The transformations were performed without (A, C, and E) or with (B, D, and F) 1.25 U of STH per ml. MDH and MR were used as the ratios 1:1 (1.25 U of each per ml) (A and B), 5:1 (6.25 U of MDH per ml and 1.25 U of MR per ml (C and D), 1:5 (1.25 U of MDH per ml and 6.25 U of MR per ml (E and F). The cofactors NADPH and NAD + were supplied at a concentration of 0.2 mM.

    Article Snippet: NADH, NADPH, NAD+ , NADP+ , and thionicotinamide NAD+ were obtained from Sigma (Poole, Dorset, United Kingdom).

    Techniques: Concentration Assay

    NAD + -dependent DHPS oxidation by recombinant dehydrogenase DhpA. The reactions contained 0.85 μg/ml DhpA in 50 mM Tris–HCl buffer at pH 9.5. The DHPS concentration was varied in the presence of 10 mM NAD + and the initial rates of NADH formation were determined spectrophotometrically as increase of absorbance at 340 nm. The data shown represents the mean ± SD of three technical replicates.

    Journal: Frontiers in Microbiology

    Article Title: Anaerobic Degradation of the Plant Sugar Sulfoquinovose Concomitant With H2S Production: Escherichia coli K-12 and Desulfovibrio sp. Strain DF1 as Co-culture Model

    doi: 10.3389/fmicb.2018.02792

    Figure Lengend Snippet: NAD + -dependent DHPS oxidation by recombinant dehydrogenase DhpA. The reactions contained 0.85 μg/ml DhpA in 50 mM Tris–HCl buffer at pH 9.5. The DHPS concentration was varied in the presence of 10 mM NAD + and the initial rates of NADH formation were determined spectrophotometrically as increase of absorbance at 340 nm. The data shown represents the mean ± SD of three technical replicates.

    Article Snippet: NAD+ was purchased from Roche, IPTG from VWR and NADH from Biomol.

    Techniques: Recombinant, Concentration Assay

    NAD + -dependent SLA oxidation to SL by recombinant dehydrogenase SlaB. Substrate SLA for candidate SLA dehydrogenase SlaB was generated from DHPS by coupling of the DhpA reaction. The reactions contained 5 μg/ml DhpA, 16.5 μg/ml SlaB, 5 mM DHPS and 10 mM NAD + in 50 mM Tris–HCl buffer at pH 9.0. Shown are HPLC-MS ion trace chromatograms (ITC) of samples taken after the reactions when screening for the quasimolecular ions of SLA (left panel) and SL (right panel). (A) Ion trace chromatograms demonstrating formation of SLA and SL in a reaction containing both enzymes, SlaB and DhpA. (B) Ion trace chromatograms of control reaction containing active DhpA and heat-inactivated SlaB. The results (A,B) were replicated once with independently prepared enzyme preparations. No quantitative measurements were possible for SLA due to the lack of an analytical standard.

    Journal: Frontiers in Microbiology

    Article Title: Anaerobic Degradation of the Plant Sugar Sulfoquinovose Concomitant With H2S Production: Escherichia coli K-12 and Desulfovibrio sp. Strain DF1 as Co-culture Model

    doi: 10.3389/fmicb.2018.02792

    Figure Lengend Snippet: NAD + -dependent SLA oxidation to SL by recombinant dehydrogenase SlaB. Substrate SLA for candidate SLA dehydrogenase SlaB was generated from DHPS by coupling of the DhpA reaction. The reactions contained 5 μg/ml DhpA, 16.5 μg/ml SlaB, 5 mM DHPS and 10 mM NAD + in 50 mM Tris–HCl buffer at pH 9.0. Shown are HPLC-MS ion trace chromatograms (ITC) of samples taken after the reactions when screening for the quasimolecular ions of SLA (left panel) and SL (right panel). (A) Ion trace chromatograms demonstrating formation of SLA and SL in a reaction containing both enzymes, SlaB and DhpA. (B) Ion trace chromatograms of control reaction containing active DhpA and heat-inactivated SlaB. The results (A,B) were replicated once with independently prepared enzyme preparations. No quantitative measurements were possible for SLA due to the lack of an analytical standard.

    Article Snippet: NAD+ was purchased from Roche, IPTG from VWR and NADH from Biomol.

    Techniques: Recombinant, Generated, High Performance Liquid Chromatography, Mass Spectrometry

    Anaerobic two-step degradation of SQ to H 2 S, as demonstrated in this study using a defined two-member bacterial co-culture. (A) Fermentation of sulfoquinovose (SQ) to 2,3-dihydoxypropane-1-sulfonate (DHPS), formate, acetate and succinate by E. coli K-12. SQ is metabolized and cleaved into DHAP and 3-sulfolactaldehyde (SLA) by a reaction sequence (enzymes YihS, V and T) analogous to the Embden-Meyerhof-Parnas pathway, as demonstrated previously for aerobic growth of E. coli ( Denger et al., 2014 ). The C 3 -organosulfonate DHPS is excreted and available as substrate to other bacteria. Under anaerobic growth conditions, E. coli funnels most of the DHAP-carbon into mixed-acid fermentation (see schematic in the gray inset on the right) to succinate, formate and acetate as fermentation products. In addition, the reduction of the SLA to DHPS, as catalyzed by the previously characterized NADH-dependent SLA reductase YihU ( Denger et al., 2014 ), serves as additional fermentation step (gray inset in the middle). (B) Fermentation of the DHPS to acetate and H 2 S by Desulfovibrio sp. strain DF1. As revealed in this study, DHPS is oxidized to 3-sulfolactate (SL) by two subsequent dehydrogenase reactions and the SL is cleaved into pyruvate and (bi)sulfite ( HSO 3 − ). The pyruvate is utilized for ATP generation concomitant with acetate excretion (and as carbon source for biomass formation, not shown). The sulfite is utilized as electron acceptor for sulfite respiration, as catalyzed by dissimilatory sulfite reductase (Dsr), and reduced to H 2 S (gray inset; for comparison, the ATP-consuming activation of sulfate is also shown). (C) The genes for DHPS-degradative enzymes identified by differential proteomics in Desulfovibrio sp. strain DF1 cells are indicated by the color coding (B,C) and/or by their IMG locus tag numbers; they are located on different contigs of the draft-genome sequence. In addition to the DHPS-desulfonation pathway genes, a candidate DHPS-transporter gene was identified by differential proteomics, i.e., for the soluble substrate binding protein (indicated in brown), which is co-encoded with candidate ABC-transporter permease and ATP-binding component genes (indicated in white), and a candidate aldehyde:ferredoxin oxidoreductase gene (oxidored.; IMG locus tag no. 130621) (see text). Other abbreviations used: SF, 6-deoxy-6-sulfofructose; SFP, 6-deoxy-6-sulfofructose phosphate; PEP, phosphoenolpyruvate; YihO, SQ importer; YihP, DHPS exporter; PFL, pyruvate-formate lyase; PFOR, pyruvate:ferredoxin oxidoreductase; Pta, phosphotransacetylase; Ack, acetate kinase; Sat, ATP sulfurylase; Apr, adenylyl-sulfate reductase.

    Journal: Frontiers in Microbiology

    Article Title: Anaerobic Degradation of the Plant Sugar Sulfoquinovose Concomitant With H2S Production: Escherichia coli K-12 and Desulfovibrio sp. Strain DF1 as Co-culture Model

    doi: 10.3389/fmicb.2018.02792

    Figure Lengend Snippet: Anaerobic two-step degradation of SQ to H 2 S, as demonstrated in this study using a defined two-member bacterial co-culture. (A) Fermentation of sulfoquinovose (SQ) to 2,3-dihydoxypropane-1-sulfonate (DHPS), formate, acetate and succinate by E. coli K-12. SQ is metabolized and cleaved into DHAP and 3-sulfolactaldehyde (SLA) by a reaction sequence (enzymes YihS, V and T) analogous to the Embden-Meyerhof-Parnas pathway, as demonstrated previously for aerobic growth of E. coli ( Denger et al., 2014 ). The C 3 -organosulfonate DHPS is excreted and available as substrate to other bacteria. Under anaerobic growth conditions, E. coli funnels most of the DHAP-carbon into mixed-acid fermentation (see schematic in the gray inset on the right) to succinate, formate and acetate as fermentation products. In addition, the reduction of the SLA to DHPS, as catalyzed by the previously characterized NADH-dependent SLA reductase YihU ( Denger et al., 2014 ), serves as additional fermentation step (gray inset in the middle). (B) Fermentation of the DHPS to acetate and H 2 S by Desulfovibrio sp. strain DF1. As revealed in this study, DHPS is oxidized to 3-sulfolactate (SL) by two subsequent dehydrogenase reactions and the SL is cleaved into pyruvate and (bi)sulfite ( HSO 3 − ). The pyruvate is utilized for ATP generation concomitant with acetate excretion (and as carbon source for biomass formation, not shown). The sulfite is utilized as electron acceptor for sulfite respiration, as catalyzed by dissimilatory sulfite reductase (Dsr), and reduced to H 2 S (gray inset; for comparison, the ATP-consuming activation of sulfate is also shown). (C) The genes for DHPS-degradative enzymes identified by differential proteomics in Desulfovibrio sp. strain DF1 cells are indicated by the color coding (B,C) and/or by their IMG locus tag numbers; they are located on different contigs of the draft-genome sequence. In addition to the DHPS-desulfonation pathway genes, a candidate DHPS-transporter gene was identified by differential proteomics, i.e., for the soluble substrate binding protein (indicated in brown), which is co-encoded with candidate ABC-transporter permease and ATP-binding component genes (indicated in white), and a candidate aldehyde:ferredoxin oxidoreductase gene (oxidored.; IMG locus tag no. 130621) (see text). Other abbreviations used: SF, 6-deoxy-6-sulfofructose; SFP, 6-deoxy-6-sulfofructose phosphate; PEP, phosphoenolpyruvate; YihO, SQ importer; YihP, DHPS exporter; PFL, pyruvate-formate lyase; PFOR, pyruvate:ferredoxin oxidoreductase; Pta, phosphotransacetylase; Ack, acetate kinase; Sat, ATP sulfurylase; Apr, adenylyl-sulfate reductase.

    Article Snippet: NAD+ was purchased from Roche, IPTG from VWR and NADH from Biomol.

    Techniques: Co-Culture Assay, Sequencing, Activation Assay, Binding Assay

    Determination of relative efficiencies of transcription initiation with NAD + vs. transcription initiation with ATP A. dsDNA transcription templates containing RNAI and T7A1 promoters (positions −40 to +3; promoter elements and transcription start sites in gray boxes); B. Representative raw data (initial RNA products of transcription reactions performed in the presence of 1 mM NAD + , 0.01–0.5 mM ATP, and [α 32 P]-CTP as extending nucleotide. C and D. Relative efficiencies of transcription initiation with NAD + vs. transcription initiation with ATP [(k cat /K M , NCIN)/(k cat /K M , ATP)]. Calculation using logarithmic regression (C; best-fit line for data points with NAD + pC/(pppApC + NAD + pC) values between 0.2 and 0.8 in (C) or non-linear regression (D); best-fit curve for data points with NAD + pC/(pppApC + NAD + pC) values between 0 and 1.

    Journal: Bio-protocol

    Article Title: RNA Capping by Transcription Initiation with Non-canonical Initiating Nucleotides (NCINs): Determination of Relative Efficiencies of Transcription Initiation with NCINs and NTPs

    doi: 10.21769/BioProtoc.2336

    Figure Lengend Snippet: Determination of relative efficiencies of transcription initiation with NAD + vs. transcription initiation with ATP A. dsDNA transcription templates containing RNAI and T7A1 promoters (positions −40 to +3; promoter elements and transcription start sites in gray boxes); B. Representative raw data (initial RNA products of transcription reactions performed in the presence of 1 mM NAD + , 0.01–0.5 mM ATP, and [α 32 P]-CTP as extending nucleotide. C and D. Relative efficiencies of transcription initiation with NAD + vs. transcription initiation with ATP [(k cat /K M , NCIN)/(k cat /K M , ATP)]. Calculation using logarithmic regression (C; best-fit line for data points with NAD + pC/(pppApC + NAD + pC) values between 0.2 and 0.8 in (C) or non-linear regression (D); best-fit curve for data points with NAD + pC/(pppApC + NAD + pC) values between 0 and 1.

    Article Snippet: NAD+ (grade I, free acid) (Roche Molecular Systems, catalog number: 10127965001) NADH (grade I, free acid) (Roche Molecular Systems, catalog number: 10107735001) Phusion Flash HF master mix (Thermo Fisher Scientific, Thermo Scientific™, catalog number: F548L) Note: For generating transcription templates .

    Techniques:

    Transcription initiation A. RNAP-promoter open complex (RPo) with unwound transcription bubble. Gray, RNAP; blue, −10-element nucleotides; i and i+1, RNAP active-center initiating nucleotide binding site and extending nucleotide binding site; boxes, DNA nucleotides (nontemplate-strand nucleotides above template-strand nucleotides). B. Structures of ATP and NAD + , Red, identical atoms in ATP and NAD + ; C. Initial RNA products formed in transcription initiation using ATP (top) or transcription initiation using NAD + (bottom). Left subpanels show initiating ATP or NAD + bound in i site; right subpanels show initial RNA products formed using CTP as extending nucleotide. Red boxes, adenosine and cytosine moieties of ATP, NAD + , and CTP; green boxes, nicotinamide-riboside moiety of NAD + .

    Journal: Bio-protocol

    Article Title: RNA Capping by Transcription Initiation with Non-canonical Initiating Nucleotides (NCINs): Determination of Relative Efficiencies of Transcription Initiation with NCINs and NTPs

    doi: 10.21769/BioProtoc.2336

    Figure Lengend Snippet: Transcription initiation A. RNAP-promoter open complex (RPo) with unwound transcription bubble. Gray, RNAP; blue, −10-element nucleotides; i and i+1, RNAP active-center initiating nucleotide binding site and extending nucleotide binding site; boxes, DNA nucleotides (nontemplate-strand nucleotides above template-strand nucleotides). B. Structures of ATP and NAD + , Red, identical atoms in ATP and NAD + ; C. Initial RNA products formed in transcription initiation using ATP (top) or transcription initiation using NAD + (bottom). Left subpanels show initiating ATP or NAD + bound in i site; right subpanels show initial RNA products formed using CTP as extending nucleotide. Red boxes, adenosine and cytosine moieties of ATP, NAD + , and CTP; green boxes, nicotinamide-riboside moiety of NAD + .

    Article Snippet: NAD+ (grade I, free acid) (Roche Molecular Systems, catalog number: 10127965001) NADH (grade I, free acid) (Roche Molecular Systems, catalog number: 10107735001) Phusion Flash HF master mix (Thermo Fisher Scientific, Thermo Scientific™, catalog number: F548L) Note: For generating transcription templates .

    Techniques: Binding Assay

    Schematic representation of the genes involved in solvent tolerance from megaplasmid pTTS12. SrpABC efflux pump is the major contributor of solvent tolerance trait from the megaplasmid pTTS12. This efflux pump is able to efficiently extrude solvents from membrane lipid bilayer. A COG5654-COG5642 family toxin-antitoxin system (SlvT and SlvA respectively) promoted the growth of P. putida S12 in the presence of low solvent concentration. In the absence of SlvA, SlvT causes toxicity by conferring cellular NAD + depletion.

    Journal: bioRxiv

    Article Title: A novel toxin-antitoxin module SlvT–SlvA governs megaplasmid stability and incites solvent tolerance in Pseudomonas putida S12

    doi: 10.1101/2020.01.02.893495

    Figure Lengend Snippet: Schematic representation of the genes involved in solvent tolerance from megaplasmid pTTS12. SrpABC efflux pump is the major contributor of solvent tolerance trait from the megaplasmid pTTS12. This efflux pump is able to efficiently extrude solvents from membrane lipid bilayer. A COG5654-COG5642 family toxin-antitoxin system (SlvT and SlvA respectively) promoted the growth of P. putida S12 in the presence of low solvent concentration. In the absence of SlvA, SlvT causes toxicity by conferring cellular NAD + depletion.

    Article Snippet: At indicated time points, NAD+ levels were measured using NAD/NADH-GloTM assay kit (Promega) according to the manufacturer’s manual.

    Techniques: Concentration Assay

    Heterologous expression of SlvAT in E. coli BL21(DE3) A. Growth curves of E. coli BL21(DE3) harbouring pBAD18-slvT and pUK21-slvA showing growth reduction after the induction of toxin by a total concentration of 0.8 % arabinose (*) and growth restoration after antitoxin induction by a total concentration of 2 mM IPTG (**). Samples were taken at the time points indicated by coloured arrows for cellular NAD + measurement. B. Flow cytometry analysis of DNA content and cell morphology visualization on E. coli BL21(DE3) during slvT and slvAT expression. Median value of green fluorescence representing DNA content during slvT expression (118.202), slvAT expression (236.056), and control (208.406) are indicated by a , b , and c respectively. Samples were taken at the time point indicated by grey arrow on figure 6A . C. Cellular NAD + measurement during the expression of toxin-antitoxin module. Induction of toxin SlvT caused a reduction in cellular NAD + level to 32.32 (±14.47) % of the control strain, while the expression of SlvA restored cellular NAD + level to 77.27 (±9.97) % of the control strain.

    Journal: bioRxiv

    Article Title: A novel toxin-antitoxin module SlvT–SlvA governs megaplasmid stability and incites solvent tolerance in Pseudomonas putida S12

    doi: 10.1101/2020.01.02.893495

    Figure Lengend Snippet: Heterologous expression of SlvAT in E. coli BL21(DE3) A. Growth curves of E. coli BL21(DE3) harbouring pBAD18-slvT and pUK21-slvA showing growth reduction after the induction of toxin by a total concentration of 0.8 % arabinose (*) and growth restoration after antitoxin induction by a total concentration of 2 mM IPTG (**). Samples were taken at the time points indicated by coloured arrows for cellular NAD + measurement. B. Flow cytometry analysis of DNA content and cell morphology visualization on E. coli BL21(DE3) during slvT and slvAT expression. Median value of green fluorescence representing DNA content during slvT expression (118.202), slvAT expression (236.056), and control (208.406) are indicated by a , b , and c respectively. Samples were taken at the time point indicated by grey arrow on figure 6A . C. Cellular NAD + measurement during the expression of toxin-antitoxin module. Induction of toxin SlvT caused a reduction in cellular NAD + level to 32.32 (±14.47) % of the control strain, while the expression of SlvA restored cellular NAD + level to 77.27 (±9.97) % of the control strain.

    Article Snippet: At indicated time points, NAD+ levels were measured using NAD/NADH-GloTM assay kit (Promega) according to the manufacturer’s manual.

    Techniques: Expressing, Concentration Assay, Flow Cytometry, Fluorescence

    TNT is a novel β-NAD + -glycohydrolase of M. tuberculosis ( a ) NAD + -glycohydrolase assay for wt TNT in the absence and presence of TNT antibody (400 ng) or IFT (100 ng).( b ) The kinetics of NAD + hydrolysis by TNT (10 nM) was determined using a fluorometric assay at pH 7.4. The values of K m and V max were determined by non-linear regression analysis using the Michaelis-Menten equation. ( c ) NAD + -glycohydrolase activity of TNT (10 nM) after heating to 65°C or 95°C for 5 minutes. The initial velocity v 0 of NAD + hydrolysis was determined at a substrate concentration of 200 μM. All data in this figure represent the means ± s.e.m. of three independent experiments.

    Journal: Nature structural & molecular biology

    Article Title: The Tuberculosis Necrotizing Toxin kills macrophages by hydrolyzing NAD

    doi: 10.1038/nsmb.3064

    Figure Lengend Snippet: TNT is a novel β-NAD + -glycohydrolase of M. tuberculosis ( a ) NAD + -glycohydrolase assay for wt TNT in the absence and presence of TNT antibody (400 ng) or IFT (100 ng).( b ) The kinetics of NAD + hydrolysis by TNT (10 nM) was determined using a fluorometric assay at pH 7.4. The values of K m and V max were determined by non-linear regression analysis using the Michaelis-Menten equation. ( c ) NAD + -glycohydrolase activity of TNT (10 nM) after heating to 65°C or 95°C for 5 minutes. The initial velocity v 0 of NAD + hydrolysis was determined at a substrate concentration of 200 μM. All data in this figure represent the means ± s.e.m. of three independent experiments.

    Article Snippet: Oligonucleotides were obtained from IDT. β-NAD+ and doxycycline was purchased from Sigma.

    Techniques: Activity Assay, Concentration Assay

    Genetic and growth characteristics displayed by dam- complemented mutant strains of UPEC relative to wild-type . (A) Dam methylation pattern in UPEC CFT073 strain subsequent to digestion with Dpn I (lane 1) and Mbo I (lane 2). The 1 kb plus DNA ladder (MW) is also shown. (B) Growth curve (CFU/milliliter versus time) for dam complement UPEC strains of CFT073, CFT073 Δ dam , cC119, and cC119 Δ dam . (C) Micrographs for wild-type (WT) and dam mutant (Δ dam ) UPEC strains, illustrating the morphological occurrence of shortened- and filamentous rods, respectively. (D) Semi-quantitative RT-PCR for mdh, rec A, and arc A expression at cycles 23, 25, and 30 for CFT073 (lanes 1–3), CFT073 Δ dam (lanes 4–6), CFT073 + pGEMdam (lanes 7–9), CFT073 Δ dam + pGEMdam (lanes 10–12). The 100 bp molecular marker MW (Promega, WI, USA) and negative control are shown (lane 13).

    Journal: Frontiers in Public Health

    Article Title: Epigenetic Influence of Dam Methylation on Gene Expression and Attachment in Uropathogenic Escherichia coli

    doi: 10.3389/fpubh.2016.00131

    Figure Lengend Snippet: Genetic and growth characteristics displayed by dam- complemented mutant strains of UPEC relative to wild-type . (A) Dam methylation pattern in UPEC CFT073 strain subsequent to digestion with Dpn I (lane 1) and Mbo I (lane 2). The 1 kb plus DNA ladder (MW) is also shown. (B) Growth curve (CFU/milliliter versus time) for dam complement UPEC strains of CFT073, CFT073 Δ dam , cC119, and cC119 Δ dam . (C) Micrographs for wild-type (WT) and dam mutant (Δ dam ) UPEC strains, illustrating the morphological occurrence of shortened- and filamentous rods, respectively. (D) Semi-quantitative RT-PCR for mdh, rec A, and arc A expression at cycles 23, 25, and 30 for CFT073 (lanes 1–3), CFT073 Δ dam (lanes 4–6), CFT073 + pGEMdam (lanes 7–9), CFT073 Δ dam + pGEMdam (lanes 10–12). The 100 bp molecular marker MW (Promega, WI, USA) and negative control are shown (lane 13).

    Article Snippet: Essentially, 0.5 μg of chromosomal and plasmid DNA was digested for 1.5 h at 37°C with 2 U Sau 3AI (Promega, WI, USA), 10 U Dpn I (New England Biolabs, MA, USA), or 2.5 U Mbo I. Sau 3AI cleaves DNA at GATC sites regardless of methylation state, Dpn I cleaves GATC sites that have a methylated adenine residue, and Mbo I cleaves unmethylated GATC sites.

    Techniques: Mutagenesis, Methylation, Quantitative RT-PCR, Expressing, Marker, Negative Control

    Phenotypic influence of Dam on P fimbriae . (A) PCR screening for pap EF in UPEC strains cC119 (lane 4), CFT073 (lane 5), and cU155 (lane 6). The 100-bp molecular weight marker (Invitrogen), negative control and positive control ( E. coli strain Lo qnr A + / pap EF + ) are represented as MW, 1 and 2, respectively. (B) PCR screening for pap I– pap B intergenic regulatory region in UPEC strains from UPEC strains cC119 (lane 2), CFT073 (lane 3), and cU155 (lane 4). The 1-kb plus molecular marker (Invitrogen, CA, USA), negative control, and positive control ( E. coli strain Lo qnr A + / pap EF + ) are represented as MW, 2 and 5, respectively. (C) Schematic representation of pSAMS1 recombinant plasmid containing cloned pap IB insert within pCRII–TOPOII vector. (D) Dam methylation patterns for pap I-B regulatory region. Sau 3AI (lane 2), Mbo I (lane 3), and Dpn I (lane 4) digests of pSAMS2 isolated from cC119 are shown. MW represents the 1 kb Plus molecular marker (Invitrogen). An undigested pap IB fragment (lane 5) is also represented. (E) Semi-quantitative (sq) RT-PCR for pap I expression in cC119 (lane 1), cC119 Δ dam (lane 2), CFT073 wild-type (lane 3) and CFT073 Δ dam (lane 4). The 1 kb Plus molecular marker (Invitrogen) and amplified chromosomal DNA for UPEC strains cC119 and CFT073 are shown in lanes MW, 5 and 6, respectively.

    Journal: Frontiers in Public Health

    Article Title: Epigenetic Influence of Dam Methylation on Gene Expression and Attachment in Uropathogenic Escherichia coli

    doi: 10.3389/fpubh.2016.00131

    Figure Lengend Snippet: Phenotypic influence of Dam on P fimbriae . (A) PCR screening for pap EF in UPEC strains cC119 (lane 4), CFT073 (lane 5), and cU155 (lane 6). The 100-bp molecular weight marker (Invitrogen), negative control and positive control ( E. coli strain Lo qnr A + / pap EF + ) are represented as MW, 1 and 2, respectively. (B) PCR screening for pap I– pap B intergenic regulatory region in UPEC strains from UPEC strains cC119 (lane 2), CFT073 (lane 3), and cU155 (lane 4). The 1-kb plus molecular marker (Invitrogen, CA, USA), negative control, and positive control ( E. coli strain Lo qnr A + / pap EF + ) are represented as MW, 2 and 5, respectively. (C) Schematic representation of pSAMS1 recombinant plasmid containing cloned pap IB insert within pCRII–TOPOII vector. (D) Dam methylation patterns for pap I-B regulatory region. Sau 3AI (lane 2), Mbo I (lane 3), and Dpn I (lane 4) digests of pSAMS2 isolated from cC119 are shown. MW represents the 1 kb Plus molecular marker (Invitrogen). An undigested pap IB fragment (lane 5) is also represented. (E) Semi-quantitative (sq) RT-PCR for pap I expression in cC119 (lane 1), cC119 Δ dam (lane 2), CFT073 wild-type (lane 3) and CFT073 Δ dam (lane 4). The 1 kb Plus molecular marker (Invitrogen) and amplified chromosomal DNA for UPEC strains cC119 and CFT073 are shown in lanes MW, 5 and 6, respectively.

    Article Snippet: Essentially, 0.5 μg of chromosomal and plasmid DNA was digested for 1.5 h at 37°C with 2 U Sau 3AI (Promega, WI, USA), 10 U Dpn I (New England Biolabs, MA, USA), or 2.5 U Mbo I. Sau 3AI cleaves DNA at GATC sites regardless of methylation state, Dpn I cleaves GATC sites that have a methylated adenine residue, and Mbo I cleaves unmethylated GATC sites.

    Techniques: Polymerase Chain Reaction, Molecular Weight, Marker, Negative Control, Positive Control, Recombinant, Plasmid Preparation, Clone Assay, Methylation, Isolation, Reverse Transcription Polymerase Chain Reaction, Expressing, Amplification

    Genotypic and growth characteristics displayed by parental and dam- mutant strains of UPEC . (A) Schematic diagram of gene disruption strategy for chromosomal insertion of chloramphenicol resistance gene from pKD3 into dam gene within UPEC chromosome subsequent to λ red recombineering with pKM208. (B) Amplified dam fragment from wild type UPEC strains CFT073 (lane 1) and cured parental strains C119 (lane 2) to produce 1071 bp amplicon. MW is 1 kb DNA ladder (Bioneer Corporation, Republic of Korea) and −ve is negative control. (C) PCR screening of UPEC candidates for dam mutation observed as 1323 bp products using primers UR427 and UR428. MW is a 1 kb Plus DNA ladder (Invitrogen, USA). (D) Dam methylation pattern in UPEC CFT073 wild type (lanes 1, 2, 8, 9, 14, 15), C119 wild type (lanes 3, 4, 10, 11, 16, 17), and E. coli K-12 substrain MG1655 (5, 12, 18) strains subsequent to digestion with Mbo I, Sau 3AI, and Dpn I. The negative control (7, 13, 19) and 1 kb Plus DNA ladder (MW) are also shown. (E) Dam methylation pattern in UPEC dam mutants CFT073 (lanes 1, 2, 3, 8, 9, 10, 15, 16, 17) and C119 wild-type (lanes 4, 5, 6, 11, 12, 13, 18, 19) subsequent to digestion with Sau 3AI, Mbo I, and Dpn I. The negative control (lanes 7, 14) and 1 kb Plus DNA ladder (MW) are also shown. (F) Growth curve (CFU/milliliter versus time) for UPEC strains CFT073, CFT073 Δ dam , cC119, and cC119 Δ dam .

    Journal: Frontiers in Public Health

    Article Title: Epigenetic Influence of Dam Methylation on Gene Expression and Attachment in Uropathogenic Escherichia coli

    doi: 10.3389/fpubh.2016.00131

    Figure Lengend Snippet: Genotypic and growth characteristics displayed by parental and dam- mutant strains of UPEC . (A) Schematic diagram of gene disruption strategy for chromosomal insertion of chloramphenicol resistance gene from pKD3 into dam gene within UPEC chromosome subsequent to λ red recombineering with pKM208. (B) Amplified dam fragment from wild type UPEC strains CFT073 (lane 1) and cured parental strains C119 (lane 2) to produce 1071 bp amplicon. MW is 1 kb DNA ladder (Bioneer Corporation, Republic of Korea) and −ve is negative control. (C) PCR screening of UPEC candidates for dam mutation observed as 1323 bp products using primers UR427 and UR428. MW is a 1 kb Plus DNA ladder (Invitrogen, USA). (D) Dam methylation pattern in UPEC CFT073 wild type (lanes 1, 2, 8, 9, 14, 15), C119 wild type (lanes 3, 4, 10, 11, 16, 17), and E. coli K-12 substrain MG1655 (5, 12, 18) strains subsequent to digestion with Mbo I, Sau 3AI, and Dpn I. The negative control (7, 13, 19) and 1 kb Plus DNA ladder (MW) are also shown. (E) Dam methylation pattern in UPEC dam mutants CFT073 (lanes 1, 2, 3, 8, 9, 10, 15, 16, 17) and C119 wild-type (lanes 4, 5, 6, 11, 12, 13, 18, 19) subsequent to digestion with Sau 3AI, Mbo I, and Dpn I. The negative control (lanes 7, 14) and 1 kb Plus DNA ladder (MW) are also shown. (F) Growth curve (CFU/milliliter versus time) for UPEC strains CFT073, CFT073 Δ dam , cC119, and cC119 Δ dam .

    Article Snippet: Essentially, 0.5 μg of chromosomal and plasmid DNA was digested for 1.5 h at 37°C with 2 U Sau 3AI (Promega, WI, USA), 10 U Dpn I (New England Biolabs, MA, USA), or 2.5 U Mbo I. Sau 3AI cleaves DNA at GATC sites regardless of methylation state, Dpn I cleaves GATC sites that have a methylated adenine residue, and Mbo I cleaves unmethylated GATC sites.

    Techniques: Mutagenesis, Amplification, Negative Control, Polymerase Chain Reaction, Methylation

    Chimera construction or insertion with a short DNA fragment . To replace a short stretch of DNA, such as a DNA fragment encoding a transmembrane region of a nAChR subunit for chimera construction, or to insert a short tag, such as FLAG-tag, into some part of a protein, amplification of the insert is not necessary. In this case, the insert can be directly included in two primers for single PCR amplification of the cDNA along with the vector. The forward primer starts immediately downstream of the insertion site and has a tail with the 3' part of the insert. The reverse primer starts immediately upstream of the insertion site and has a tail with 5' part of the insert. Two primers only require ~16-base overlap. Thus, for a 120 bp insertion, each primer needs to have a 68-base tail for insertion and ~17-22 bases for annealing (depending on the GC content). The total length of each primer will be about 85-90 bases. Dpn I digestion and transformation are the same as in Figure 1.

    Journal: BMC Biotechnology

    Article Title: FastCloning: a highly simplified, purification-free, sequence- and ligation-independent PCR cloning method

    doi: 10.1186/1472-6750-11-92

    Figure Lengend Snippet: Chimera construction or insertion with a short DNA fragment . To replace a short stretch of DNA, such as a DNA fragment encoding a transmembrane region of a nAChR subunit for chimera construction, or to insert a short tag, such as FLAG-tag, into some part of a protein, amplification of the insert is not necessary. In this case, the insert can be directly included in two primers for single PCR amplification of the cDNA along with the vector. The forward primer starts immediately downstream of the insertion site and has a tail with the 3' part of the insert. The reverse primer starts immediately upstream of the insertion site and has a tail with 5' part of the insert. Two primers only require ~16-base overlap. Thus, for a 120 bp insertion, each primer needs to have a 68-base tail for insertion and ~17-22 bases for annealing (depending on the GC content). The total length of each primer will be about 85-90 bases. Dpn I digestion and transformation are the same as in Figure 1.

    Article Snippet: After confirmation of PCR products, 1 μl of Dpn I enzyme (New England Biolabs) was added into the remaining unpurified PCR reactions (45 μl for each product) for vector or insert separately.

    Techniques: FLAG-tag, Amplification, Polymerase Chain Reaction, Plasmid Preparation, Transformation Assay

    Optimization of cloning conditions . (A) PCR amplification of a target cDNA (human nAChR α9 subunit) and the pGEMHE vector using different DNA polymerases: Pfu Turbo, PfuUltra and Phusion. (B) Comparison of number of colonies grown on the plates after transformation. Three different vector-to-insert ratios (1:1, 1:2, and 1:4) during Dpn I digestion and three amounts of vector-insert mixtures (2, 4, and 8 μl) for transformation were tested. See text for details. (C) Clone validation by PCR using GoTaq DNA polymerase. Lanes 1 to 12: target clones to be validated; Lane 13: 1 Kb plus DNA ladder; Lane 14: pGEMHE vector control; Lane 15: negative control using pCR4-TOPO-α9 parent plasmid. (D) Clone validation by restriction digestion to exclude unusual constructs. Lane 1: 1 Kb plus DNA ladder, Lanes 2-11: target clones double digested with Kpn I and Nhe I. Note that this digestion resulted in a pGEM vector and an insert with α9 nAChR plus the 5'UTR and 3'UTR of Xenopus β-globin.

    Journal: BMC Biotechnology

    Article Title: FastCloning: a highly simplified, purification-free, sequence- and ligation-independent PCR cloning method

    doi: 10.1186/1472-6750-11-92

    Figure Lengend Snippet: Optimization of cloning conditions . (A) PCR amplification of a target cDNA (human nAChR α9 subunit) and the pGEMHE vector using different DNA polymerases: Pfu Turbo, PfuUltra and Phusion. (B) Comparison of number of colonies grown on the plates after transformation. Three different vector-to-insert ratios (1:1, 1:2, and 1:4) during Dpn I digestion and three amounts of vector-insert mixtures (2, 4, and 8 μl) for transformation were tested. See text for details. (C) Clone validation by PCR using GoTaq DNA polymerase. Lanes 1 to 12: target clones to be validated; Lane 13: 1 Kb plus DNA ladder; Lane 14: pGEMHE vector control; Lane 15: negative control using pCR4-TOPO-α9 parent plasmid. (D) Clone validation by restriction digestion to exclude unusual constructs. Lane 1: 1 Kb plus DNA ladder, Lanes 2-11: target clones double digested with Kpn I and Nhe I. Note that this digestion resulted in a pGEM vector and an insert with α9 nAChR plus the 5'UTR and 3'UTR of Xenopus β-globin.

    Article Snippet: After confirmation of PCR products, 1 μl of Dpn I enzyme (New England Biolabs) was added into the remaining unpurified PCR reactions (45 μl for each product) for vector or insert separately.

    Techniques: Clone Assay, Polymerase Chain Reaction, Amplification, Plasmid Preparation, Transformation Assay, Negative Control, Construct

    The procedures for FastCloning: Step 1. PCR amplification of vector and insert. Note that the primer pair for insert amplification has 16-base tails overlapping with the PCR-amplified vector ends. Step 2. Dpn I digestion. The parent DNA templates (if in a plasmid) for PCR amplification needs to be methylated in order to be compatible to Dpn I digestion. Although the detailed mechanism is not known, it is likely that the 3' exonuclease activity of the high fidelity DNA polymerase directly creates sticky ends for the overlapped regions of the vector and insert during Dpn I digestion, allowing them to form a circular construct with nicks. Step 3. transformation into competent E. coli . cells. The nicks will be repaired after transformation into the bacteria.

    Journal: BMC Biotechnology

    Article Title: FastCloning: a highly simplified, purification-free, sequence- and ligation-independent PCR cloning method

    doi: 10.1186/1472-6750-11-92

    Figure Lengend Snippet: The procedures for FastCloning: Step 1. PCR amplification of vector and insert. Note that the primer pair for insert amplification has 16-base tails overlapping with the PCR-amplified vector ends. Step 2. Dpn I digestion. The parent DNA templates (if in a plasmid) for PCR amplification needs to be methylated in order to be compatible to Dpn I digestion. Although the detailed mechanism is not known, it is likely that the 3' exonuclease activity of the high fidelity DNA polymerase directly creates sticky ends for the overlapped regions of the vector and insert during Dpn I digestion, allowing them to form a circular construct with nicks. Step 3. transformation into competent E. coli . cells. The nicks will be repaired after transformation into the bacteria.

    Article Snippet: After confirmation of PCR products, 1 μl of Dpn I enzyme (New England Biolabs) was added into the remaining unpurified PCR reactions (45 μl for each product) for vector or insert separately.

    Techniques: Polymerase Chain Reaction, Amplification, Plasmid Preparation, Methylation, Activity Assay, Construct, Transformation Assay