Structured Review

Toyobo high fidelity dna polymerase
Typical mass spectra of four <t>biomarker</t> proteins in E. coli . A) MALDI mass spectra of non-EHEC E. coli strain NBRC12713 and EHEC E. coli strain O157 GTC 14513. Three biomarker peaks, HdeB ( m/z 9066.2 [M+H] + ), ribosomal protein S15 ( m/z 10138.6/10166.6 [M+H] + ) and L25 ( m/z 10676.4/10694.4 [M+H] + ), measured using sinapic acid as the matrix, are shown. B) Biomarker peak of <t>DNA</t> binding protein H-NS ( m/z 15409.4/15425.4 [M+H] + ) for strains O26, O111 and other E. coli .
High Fidelity Dna Polymerase, supplied by Toyobo, used in various techniques. Bioz Stars score: 93/100, based on 43 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Images

1) Product Images from "Discrimination of Escherichia coli O157, O26 and O111 from Other Serovars by MALDI-TOF MS Based on the S10-GERMS Method"

Article Title: Discrimination of Escherichia coli O157, O26 and O111 from Other Serovars by MALDI-TOF MS Based on the S10-GERMS Method

Journal: PLoS ONE

doi: 10.1371/journal.pone.0113458

Typical mass spectra of four biomarker proteins in E. coli . A) MALDI mass spectra of non-EHEC E. coli strain NBRC12713 and EHEC E. coli strain O157 GTC 14513. Three biomarker peaks, HdeB ( m/z 9066.2 [M+H] + ), ribosomal protein S15 ( m/z 10138.6/10166.6 [M+H] + ) and L25 ( m/z 10676.4/10694.4 [M+H] + ), measured using sinapic acid as the matrix, are shown. B) Biomarker peak of DNA binding protein H-NS ( m/z 15409.4/15425.4 [M+H] + ) for strains O26, O111 and other E. coli .
Figure Legend Snippet: Typical mass spectra of four biomarker proteins in E. coli . A) MALDI mass spectra of non-EHEC E. coli strain NBRC12713 and EHEC E. coli strain O157 GTC 14513. Three biomarker peaks, HdeB ( m/z 9066.2 [M+H] + ), ribosomal protein S15 ( m/z 10138.6/10166.6 [M+H] + ) and L25 ( m/z 10676.4/10694.4 [M+H] + ), measured using sinapic acid as the matrix, are shown. B) Biomarker peak of DNA binding protein H-NS ( m/z 15409.4/15425.4 [M+H] + ) for strains O26, O111 and other E. coli .

Techniques Used: Biomarker Assay, Binding Assay

2) Product Images from "A Novel TetR Family Transcriptional Regulator, CalR3, Negatively Controls Calcimycin Biosynthesis in Streptomyces chartreusis NRRL 3882"

Article Title: A Novel TetR Family Transcriptional Regulator, CalR3, Negatively Controls Calcimycin Biosynthesis in Streptomyces chartreusis NRRL 3882

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2017.02371

Determination of the binding sites of CalR3 protein. (A) DNase I footprinting assay of CalR3 on the T-R3L region. The fluorograms correspond to the control reaction without CalR3 protein and to the protection patterns with increasing CalR3 protein (0.12 and 0.24 μg). (B) Nucleotide sequences of the T-R3L promoter region and CalR3-binding site. Solid line, CalR3-binding site; shaded areas, translational start codons; bent arrow, TSPs; boxes, putative –10 and –35 regions. (C) Mutations introduced into the CalR3 binding sites I and II regions. Each probe was 80 bp. Probe m0 was WT DNA containing a 14 bp palindromic sequence in site II. Base substitutions were introduced into probe m0 to produce mutated probes m1, m2, and m1 2, respectively. Altered nucleotides are underlined. (D) EMSAs with WT DNA probe and mutated probes. Each reaction mixture contained 1.5 μM probe and 0.8 μM of purified His 6 -CalR3 protein.
Figure Legend Snippet: Determination of the binding sites of CalR3 protein. (A) DNase I footprinting assay of CalR3 on the T-R3L region. The fluorograms correspond to the control reaction without CalR3 protein and to the protection patterns with increasing CalR3 protein (0.12 and 0.24 μg). (B) Nucleotide sequences of the T-R3L promoter region and CalR3-binding site. Solid line, CalR3-binding site; shaded areas, translational start codons; bent arrow, TSPs; boxes, putative –10 and –35 regions. (C) Mutations introduced into the CalR3 binding sites I and II regions. Each probe was 80 bp. Probe m0 was WT DNA containing a 14 bp palindromic sequence in site II. Base substitutions were introduced into probe m0 to produce mutated probes m1, m2, and m1 2, respectively. Altered nucleotides are underlined. (D) EMSAs with WT DNA probe and mutated probes. Each reaction mixture contained 1.5 μM probe and 0.8 μM of purified His 6 -CalR3 protein.

Techniques Used: Binding Assay, Footprinting, Sequencing, Purification

3) Product Images from "Concurrent Overexpression of OsGS1;1 and OsGS2 Genes in Transgenic Rice (Oryza sativa L.): Impact on Tolerance to Abiotic Stresses"

Article Title: Concurrent Overexpression of OsGS1;1 and OsGS2 Genes in Transgenic Rice (Oryza sativa L.): Impact on Tolerance to Abiotic Stresses

Journal: Frontiers in Plant Science

doi: 10.3389/fpls.2018.00786

Molecular and biochemical analysis of transgenic rice lines co-overexpressing OsGS1;1 and OsGS2 . (A) PCR amplification of hygromycin phosphotransferase ( hpt ), OsGS1;1 and OSGS2 genes using specific primers in wild type ( wt ), null segregant ( ns ), and five positive T 2 transgenic lines (L1-L5). M: 1Kb DNA ladder (+): positive PCR control (pMDC99) and (–) water blank. (B) Southern blot analysis of wt and five T 2 transgenic lines (L1, L2, L3, L4, and L5), probed with hpt gene probe showing single copy insertion. (C) Semi quantitative RT-PCR showing overexpression of OsGS1;1 and OsGS2 in transgenic lines (L1, L4, and L5) as compared to wt . The rice eEF1α gene was used as a reference gene and rRNA was used as loading control. (D ; top panel) Immunoblot analysis of three transgenic rice lines (L1, L4, and L5) and wt using a recombinant antibody which detects both OsGS1;1 and OsGS2 isoforms (black lines separate spliced regions from same blot) ( D ; bottom panel) Coomassie blue stained Rubisco large subunit (RubL) was used as loading control. (E) Total GS activity of three transgenic rice lines (L1, L4, and L5) in comparison to wt ). One unit of GS activity represents 1.0 μmol of γ-glutamylhydroxamate produced in 20 min. Asterisks above bars indicate significant differences from wt (* at p ≤ 0.05 and ** at p ≤ 0.01).
Figure Legend Snippet: Molecular and biochemical analysis of transgenic rice lines co-overexpressing OsGS1;1 and OsGS2 . (A) PCR amplification of hygromycin phosphotransferase ( hpt ), OsGS1;1 and OSGS2 genes using specific primers in wild type ( wt ), null segregant ( ns ), and five positive T 2 transgenic lines (L1-L5). M: 1Kb DNA ladder (+): positive PCR control (pMDC99) and (–) water blank. (B) Southern blot analysis of wt and five T 2 transgenic lines (L1, L2, L3, L4, and L5), probed with hpt gene probe showing single copy insertion. (C) Semi quantitative RT-PCR showing overexpression of OsGS1;1 and OsGS2 in transgenic lines (L1, L4, and L5) as compared to wt . The rice eEF1α gene was used as a reference gene and rRNA was used as loading control. (D ; top panel) Immunoblot analysis of three transgenic rice lines (L1, L4, and L5) and wt using a recombinant antibody which detects both OsGS1;1 and OsGS2 isoforms (black lines separate spliced regions from same blot) ( D ; bottom panel) Coomassie blue stained Rubisco large subunit (RubL) was used as loading control. (E) Total GS activity of three transgenic rice lines (L1, L4, and L5) in comparison to wt ). One unit of GS activity represents 1.0 μmol of γ-glutamylhydroxamate produced in 20 min. Asterisks above bars indicate significant differences from wt (* at p ≤ 0.05 and ** at p ≤ 0.01).

Techniques Used: Transgenic Assay, Polymerase Chain Reaction, Amplification, Southern Blot, Quantitative RT-PCR, Over Expression, Recombinant, Staining, Activity Assay, Produced

4) Product Images from "Streptomyces lividans Blasticidin S Deaminase and Its Application in Engineering a Blasticidin S-Producing Strain for Ease of Genetic Manipulation"

Article Title: Streptomyces lividans Blasticidin S Deaminase and Its Application in Engineering a Blasticidin S-Producing Strain for Ease of Genetic Manipulation

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.03254-12

High-performance liquid chromatography analysis of the metabolite production of S. lividans WJ2. (A) Confirmation of the genotype of the SLBSD mutant WJ2 by PCR with primer Con-BKO (see Table S2 in the supplemental material). M, DNA marker; HXY16, PCR product targeting the SLBSD gene from S. lividans HXY16; WJ2, PCR product targeting the SLBSD gene from S. lividans WJ2. (B) High-performance liquid chromatography comparison of the blasticidin S standard to the purified fermentation broth of S. lividans WJ2 and S. lividans HXY16.
Figure Legend Snippet: High-performance liquid chromatography analysis of the metabolite production of S. lividans WJ2. (A) Confirmation of the genotype of the SLBSD mutant WJ2 by PCR with primer Con-BKO (see Table S2 in the supplemental material). M, DNA marker; HXY16, PCR product targeting the SLBSD gene from S. lividans HXY16; WJ2, PCR product targeting the SLBSD gene from S. lividans WJ2. (B) High-performance liquid chromatography comparison of the blasticidin S standard to the purified fermentation broth of S. lividans WJ2 and S. lividans HXY16.

Techniques Used: High Performance Liquid Chromatography, Mutagenesis, Polymerase Chain Reaction, Marker, Purification

Assay of the blasticidin S production of the Δ blsF and Δ blsE mutant strains, S. lividans WJ4 and S. lividans WJ5, respectively. (A) Confirmation of the Δ blsF mutant WJ4 by PCR with primer Con-blsF (see Table S2 in the supplemental material). M, DNA marker; WJ2, PCR product targeting blsF from S. lividans WJ2; WJ4, PCR product targeting blsF from S. lividans WJ4. (B) High-performance liquid chromatography comparison of the purified fermentation broth of WJ4 to that of WJ2. (C) Confirmation of the Δ blsE mutant WJ5 by PCR with primer Con-blsE (see Table S2). M, DNA marker; WJ2, PCR product targeting blsE from S. lividans WJ2; WJ5, PCR product targeting blsE from S. lividans WJ5. (D) High-performance liquid chromatography comparison of the untreated fermentation broth of WJ5 to that of WJ2 and HXY16.
Figure Legend Snippet: Assay of the blasticidin S production of the Δ blsF and Δ blsE mutant strains, S. lividans WJ4 and S. lividans WJ5, respectively. (A) Confirmation of the Δ blsF mutant WJ4 by PCR with primer Con-blsF (see Table S2 in the supplemental material). M, DNA marker; WJ2, PCR product targeting blsF from S. lividans WJ2; WJ4, PCR product targeting blsF from S. lividans WJ4. (B) High-performance liquid chromatography comparison of the purified fermentation broth of WJ4 to that of WJ2. (C) Confirmation of the Δ blsE mutant WJ5 by PCR with primer Con-blsE (see Table S2). M, DNA marker; WJ2, PCR product targeting blsE from S. lividans WJ2; WJ5, PCR product targeting blsE from S. lividans WJ5. (D) High-performance liquid chromatography comparison of the untreated fermentation broth of WJ5 to that of WJ2 and HXY16.

Techniques Used: Mutagenesis, Polymerase Chain Reaction, Marker, High Performance Liquid Chromatography, Purification

Assay of the blasticidin S production of the Δ blsL mutant strain S. lividans WJ3. (A) High-performance liquid chromatography comparison of the blasticidin S standard to the purified fermentation broth of WJ3, WJ2, and HXY16. LC-MS analysis revealed the peak presented in the fermentation broth of LL2 and WJ2, with a dominant peak at m/z 409. (B) MS/MS analysis of the m/z 409 parent ion. Each labeled daughter peak (corresponding structures are shown in the inset) could be derived from demethylblasticidin S. (C) PCR confirmation of the genotype of the Δ blsL mutant WJ3 with primer Con-blsL (see Table S2 in the supplemental material). M, DNA marker; WJ2, PCR product targeting blsL from S. lividans WJ2; WJ3, PCR product targeting blsL from S. lividans WJ3.
Figure Legend Snippet: Assay of the blasticidin S production of the Δ blsL mutant strain S. lividans WJ3. (A) High-performance liquid chromatography comparison of the blasticidin S standard to the purified fermentation broth of WJ3, WJ2, and HXY16. LC-MS analysis revealed the peak presented in the fermentation broth of LL2 and WJ2, with a dominant peak at m/z 409. (B) MS/MS analysis of the m/z 409 parent ion. Each labeled daughter peak (corresponding structures are shown in the inset) could be derived from demethylblasticidin S. (C) PCR confirmation of the genotype of the Δ blsL mutant WJ3 with primer Con-blsL (see Table S2 in the supplemental material). M, DNA marker; WJ2, PCR product targeting blsL from S. lividans WJ2; WJ3, PCR product targeting blsL from S. lividans WJ3.

Techniques Used: Mutagenesis, High Performance Liquid Chromatography, Purification, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Labeling, Derivative Assay, Polymerase Chain Reaction, Marker

5) Product Images from "Concurrent Overexpression of OsGS1;1 and OsGS2 Genes in Transgenic Rice (Oryza sativa L.): Impact on Tolerance to Abiotic Stresses"

Article Title: Concurrent Overexpression of OsGS1;1 and OsGS2 Genes in Transgenic Rice (Oryza sativa L.): Impact on Tolerance to Abiotic Stresses

Journal: Frontiers in Plant Science

doi: 10.3389/fpls.2018.00786

Molecular and biochemical analysis of transgenic rice lines co-overexpressing OsGS1;1 and OsGS2 . (A) PCR amplification of hygromycin phosphotransferase ( hpt ), OsGS1;1 and OSGS2 genes using specific primers in wild type ( wt ), null segregant ( ns ), and five positive T 2 transgenic lines (L1-L5). M: 1Kb DNA ladder (+): positive PCR control (pMDC99) and (–) water blank. (B) Southern blot analysis of wt and five T 2 transgenic lines (L1, L2, L3, L4, and L5), probed with hpt gene probe showing single copy insertion. (C) Semi quantitative RT-PCR showing overexpression of OsGS1;1 and OsGS2 in transgenic lines (L1, L4, and L5) as compared to wt . The rice eEF1α gene was used as a reference gene and rRNA was used as loading control. (D ; top panel) Immunoblot analysis of three transgenic rice lines (L1, L4, and L5) and wt using a recombinant antibody which detects both OsGS1;1 and OsGS2 isoforms (black lines separate spliced regions from same blot) ( D ; bottom panel) Coomassie blue stained Rubisco large subunit (RubL) was used as loading control. (E) Total GS activity of three transgenic rice lines (L1, L4, and L5) in comparison to wt as assayed by a modified semi-biosynthetic assay (Singh and Ghosh, 2013 ). One unit of GS activity represents 1.0 μmol of γ-glutamylhydroxamate produced in 20 min. Asterisks above bars indicate significant differences from wt (* at p ≤ 0.05 and ** at p ≤ 0.01).
Figure Legend Snippet: Molecular and biochemical analysis of transgenic rice lines co-overexpressing OsGS1;1 and OsGS2 . (A) PCR amplification of hygromycin phosphotransferase ( hpt ), OsGS1;1 and OSGS2 genes using specific primers in wild type ( wt ), null segregant ( ns ), and five positive T 2 transgenic lines (L1-L5). M: 1Kb DNA ladder (+): positive PCR control (pMDC99) and (–) water blank. (B) Southern blot analysis of wt and five T 2 transgenic lines (L1, L2, L3, L4, and L5), probed with hpt gene probe showing single copy insertion. (C) Semi quantitative RT-PCR showing overexpression of OsGS1;1 and OsGS2 in transgenic lines (L1, L4, and L5) as compared to wt . The rice eEF1α gene was used as a reference gene and rRNA was used as loading control. (D ; top panel) Immunoblot analysis of three transgenic rice lines (L1, L4, and L5) and wt using a recombinant antibody which detects both OsGS1;1 and OsGS2 isoforms (black lines separate spliced regions from same blot) ( D ; bottom panel) Coomassie blue stained Rubisco large subunit (RubL) was used as loading control. (E) Total GS activity of three transgenic rice lines (L1, L4, and L5) in comparison to wt as assayed by a modified semi-biosynthetic assay (Singh and Ghosh, 2013 ). One unit of GS activity represents 1.0 μmol of γ-glutamylhydroxamate produced in 20 min. Asterisks above bars indicate significant differences from wt (* at p ≤ 0.05 and ** at p ≤ 0.01).

Techniques Used: Transgenic Assay, Polymerase Chain Reaction, Amplification, Southern Blot, Quantitative RT-PCR, Over Expression, Recombinant, Staining, Activity Assay, Modification, Produced

Related Articles

Clone Assay:

Article Title: Concurrent Overexpression of OsGS1;1 and OsGS2 Genes in Transgenic Rice (Oryza sativa L.): Impact on Tolerance to Abiotic Stresses
Article Snippet: .. The full-length coding sequences of OsGS1;1 (1113 bp) and OsGS2 (1287 bp) were amplified by PCR from rice (Oryza sativa L. ssp japonica cv Nipponbare) cDNA using a high fidelity DNA polymerase (KOD plus, Toyobo, Japan) and cloned into pCR-4-TOPO vector (Invitrogen, USA). .. Following verification by sequencing, the OsGS1;1 gene was sub-cloned into a Gateway compatible entry vector EV-1 (pL12R34-Ap) in between the rice Actin 2 (OsAct2 ) promoter and rice Actin 2 (OsAct2 ) terminator, whereas the OsGS2 gene was cloned into the Gateway compatible EV-2 (pL34R12-Cm-ccdB) vector under the rice Actin 1 (OsAct1 ) promoter and rice Actin 1 (OsAct1 ) terminator.

Article Title: Streptomyces lividans Blasticidin S Deaminase and Its Application in Engineering a Blasticidin S-Producing Strain for Ease of Genetic Manipulation
Article Snippet: .. PCR was carried out with high-fidelity DNA polymerase (KOD-Plus; Toyobo), and products were gel purified, digested with the appropriate enzymes, and cloned into the corresponding restriction sites of the pET28a(+) vector (Novagen). .. The resulting plasmid, named pWJ4, was used to transform E. coli DH10B for sequencing.

Article Title: Concurrent Overexpression of OsGS1;1 and OsGS2 Genes in Transgenic Rice (Oryza sativa L.): Impact on Tolerance to Abiotic Stresses
Article Snippet: .. The full-length coding sequences of OsGS1;1 (1113 bp) and OsGS2 (1287 bp) were amplified by PCR from rice ( Oryza sativa L. ssp japonica cv Nipponbare) cDNA using a high fidelity DNA polymerase (KOD plus, Toyobo, Japan) and cloned into pCR-4-TOPO vector (Invitrogen, USA). .. Following verification by sequencing, the OsGS1;1 gene was sub-cloned into a Gateway compatible entry vector EV-1 (pL12R34-Ap) in between the rice Actin 2 ( OsAct2 ) promoter and rice Actin 2 ( OsAct2 ) terminator, whereas the OsGS2 gene was cloned into the Gateway compatible EV-2 (pL34R12-Cm-ccdB) vector under the rice Actin 1 ( OsAct1 ) promoter and rice Actin 1 ( OsAct1 ) terminator.

Amplification:

Article Title: Concurrent Overexpression of OsGS1;1 and OsGS2 Genes in Transgenic Rice (Oryza sativa L.): Impact on Tolerance to Abiotic Stresses
Article Snippet: .. The full-length coding sequences of OsGS1;1 (1113 bp) and OsGS2 (1287 bp) were amplified by PCR from rice (Oryza sativa L. ssp japonica cv Nipponbare) cDNA using a high fidelity DNA polymerase (KOD plus, Toyobo, Japan) and cloned into pCR-4-TOPO vector (Invitrogen, USA). .. Following verification by sequencing, the OsGS1;1 gene was sub-cloned into a Gateway compatible entry vector EV-1 (pL12R34-Ap) in between the rice Actin 2 (OsAct2 ) promoter and rice Actin 2 (OsAct2 ) terminator, whereas the OsGS2 gene was cloned into the Gateway compatible EV-2 (pL34R12-Cm-ccdB) vector under the rice Actin 1 (OsAct1 ) promoter and rice Actin 1 (OsAct1 ) terminator.

Article Title: Discrimination of Escherichia coli O157, O26 and O111 from Other Serovars by MALDI-TOF MS Based on the S10-GERMS Method
Article Snippet: .. In brief, respective regions of ribosomal protein-encoding genes (≈5 kbp) or genes encoding biomarker proteins were amplified using high-fidelity DNA polymerase, KOD plus (Toyobo, Osaka, Japan), and primers designed against the consensus DNA sequences up- and down-stream of the target regions in the E. coli genome sequences in the NCBI database. .. Sequencing reactions were carried out using a BigDye ver.

Article Title: A Novel TetR Family Transcriptional Regulator, CalR3, Negatively Controls Calcimycin Biosynthesis in Streptomyces chartreusis NRRL 3882
Article Snippet: .. For the complementation of strain GLX26(ΔcalR3 ), the intact calR3 gene was amplified from S. chartreusis NRRL 3882 genomic DNA with the primers calR3-F5 and calR3-F6 using a high-fidelity DNA polymerase (KOD-plus, TOYOBO). .. The resulting PCR fragment was cloned into an integrative plasmid, pJTU2170, that was derived from plasmid pIB139 , resulting in pJTU3795.

Article Title: A Novel Member of the Trehalose Transporter Family Functions as an H+-Dependent Trehalose Transporter in the Reabsorption of Trehalose in Malpighian Tubules
Article Snippet: .. The template DNA for cRNA synthesis was amplified with high-fidelity DNA polymerase (KOD plus, TOYOBO, Osaka, Japan) from the expression vectors by using primers containing the T7 and T3 promoters. .. The Nlst8 cRNAs were obtained by using the mMESSAGE mMACHINE T7 kit (Life Technologies) according to the manufacturer’s standard protocols.

Article Title: Concurrent Overexpression of OsGS1;1 and OsGS2 Genes in Transgenic Rice (Oryza sativa L.): Impact on Tolerance to Abiotic Stresses
Article Snippet: .. The full-length coding sequences of OsGS1;1 (1113 bp) and OsGS2 (1287 bp) were amplified by PCR from rice ( Oryza sativa L. ssp japonica cv Nipponbare) cDNA using a high fidelity DNA polymerase (KOD plus, Toyobo, Japan) and cloned into pCR-4-TOPO vector (Invitrogen, USA). .. Following verification by sequencing, the OsGS1;1 gene was sub-cloned into a Gateway compatible entry vector EV-1 (pL12R34-Ap) in between the rice Actin 2 ( OsAct2 ) promoter and rice Actin 2 ( OsAct2 ) terminator, whereas the OsGS2 gene was cloned into the Gateway compatible EV-2 (pL34R12-Cm-ccdB) vector under the rice Actin 1 ( OsAct1 ) promoter and rice Actin 1 ( OsAct1 ) terminator.

Mutagenesis:

Article Title: Na+/H+ Antiport Is Essential for Yersinia pestis Virulence
Article Snippet: .. Because nhaB from KIM5-3001 ( nhaB ) contained a frameshift mutation, we performed site-directed mutagenesis to restore the function of the gene by using primers nhaB-pBAD-SDM-P1 and nhaB-pBAD-SDM-P2 , a high-fidelity DNA polymerase, KOD -Plus-, version 2 (Toyobo), and DpnI (Invitrogen). .. The site-directed mutagenesis was confirmed by sequencing.

Biomarker Assay:

Article Title: Discrimination of Escherichia coli O157, O26 and O111 from Other Serovars by MALDI-TOF MS Based on the S10-GERMS Method
Article Snippet: .. In brief, respective regions of ribosomal protein-encoding genes (≈5 kbp) or genes encoding biomarker proteins were amplified using high-fidelity DNA polymerase, KOD plus (Toyobo, Osaka, Japan), and primers designed against the consensus DNA sequences up- and down-stream of the target regions in the E. coli genome sequences in the NCBI database. .. Sequencing reactions were carried out using a BigDye ver.

Purification:

Article Title: Streptomyces lividans Blasticidin S Deaminase and Its Application in Engineering a Blasticidin S-Producing Strain for Ease of Genetic Manipulation
Article Snippet: .. PCR was carried out with high-fidelity DNA polymerase (KOD-Plus; Toyobo), and products were gel purified, digested with the appropriate enzymes, and cloned into the corresponding restriction sites of the pET28a(+) vector (Novagen). .. The resulting plasmid, named pWJ4, was used to transform E. coli DH10B for sequencing.

Expressing:

Article Title: A Novel Member of the Trehalose Transporter Family Functions as an H+-Dependent Trehalose Transporter in the Reabsorption of Trehalose in Malpighian Tubules
Article Snippet: .. The template DNA for cRNA synthesis was amplified with high-fidelity DNA polymerase (KOD plus, TOYOBO, Osaka, Japan) from the expression vectors by using primers containing the T7 and T3 promoters. .. The Nlst8 cRNAs were obtained by using the mMESSAGE mMACHINE T7 kit (Life Technologies) according to the manufacturer’s standard protocols.

Polymerase Chain Reaction:

Article Title: Concurrent Overexpression of OsGS1;1 and OsGS2 Genes in Transgenic Rice (Oryza sativa L.): Impact on Tolerance to Abiotic Stresses
Article Snippet: .. The full-length coding sequences of OsGS1;1 (1113 bp) and OsGS2 (1287 bp) were amplified by PCR from rice (Oryza sativa L. ssp japonica cv Nipponbare) cDNA using a high fidelity DNA polymerase (KOD plus, Toyobo, Japan) and cloned into pCR-4-TOPO vector (Invitrogen, USA). .. Following verification by sequencing, the OsGS1;1 gene was sub-cloned into a Gateway compatible entry vector EV-1 (pL12R34-Ap) in between the rice Actin 2 (OsAct2 ) promoter and rice Actin 2 (OsAct2 ) terminator, whereas the OsGS2 gene was cloned into the Gateway compatible EV-2 (pL34R12-Cm-ccdB) vector under the rice Actin 1 (OsAct1 ) promoter and rice Actin 1 (OsAct1 ) terminator.

Article Title: Streptomyces lividans Blasticidin S Deaminase and Its Application in Engineering a Blasticidin S-Producing Strain for Ease of Genetic Manipulation
Article Snippet: .. PCR was carried out with high-fidelity DNA polymerase (KOD-Plus; Toyobo), and products were gel purified, digested with the appropriate enzymes, and cloned into the corresponding restriction sites of the pET28a(+) vector (Novagen). .. The resulting plasmid, named pWJ4, was used to transform E. coli DH10B for sequencing.

Article Title: Isolation of a Multiheme Protein with Features of a Hydrazine-Oxidizing Enzyme from an Anaerobic Ammonium-Oxidizing Enrichment Culture ▿
Article Snippet: .. All PCR procedures were performed using a high-fidelity DNA polymerase, KOD-Plus (TOYOBO Co., Ltd., Osaka, Japan). .. The fragment was labeled with digoxigenin and used as a probe in a Southern analysis of extracted metagenomic DNA to select restriction enzymes suitable for inverse PCR.

Article Title: Concurrent Overexpression of OsGS1;1 and OsGS2 Genes in Transgenic Rice (Oryza sativa L.): Impact on Tolerance to Abiotic Stresses
Article Snippet: .. The full-length coding sequences of OsGS1;1 (1113 bp) and OsGS2 (1287 bp) were amplified by PCR from rice ( Oryza sativa L. ssp japonica cv Nipponbare) cDNA using a high fidelity DNA polymerase (KOD plus, Toyobo, Japan) and cloned into pCR-4-TOPO vector (Invitrogen, USA). .. Following verification by sequencing, the OsGS1;1 gene was sub-cloned into a Gateway compatible entry vector EV-1 (pL12R34-Ap) in between the rice Actin 2 ( OsAct2 ) promoter and rice Actin 2 ( OsAct2 ) terminator, whereas the OsGS2 gene was cloned into the Gateway compatible EV-2 (pL34R12-Cm-ccdB) vector under the rice Actin 1 ( OsAct1 ) promoter and rice Actin 1 ( OsAct1 ) terminator.

Plasmid Preparation:

Article Title: Concurrent Overexpression of OsGS1;1 and OsGS2 Genes in Transgenic Rice (Oryza sativa L.): Impact on Tolerance to Abiotic Stresses
Article Snippet: .. The full-length coding sequences of OsGS1;1 (1113 bp) and OsGS2 (1287 bp) were amplified by PCR from rice (Oryza sativa L. ssp japonica cv Nipponbare) cDNA using a high fidelity DNA polymerase (KOD plus, Toyobo, Japan) and cloned into pCR-4-TOPO vector (Invitrogen, USA). .. Following verification by sequencing, the OsGS1;1 gene was sub-cloned into a Gateway compatible entry vector EV-1 (pL12R34-Ap) in between the rice Actin 2 (OsAct2 ) promoter and rice Actin 2 (OsAct2 ) terminator, whereas the OsGS2 gene was cloned into the Gateway compatible EV-2 (pL34R12-Cm-ccdB) vector under the rice Actin 1 (OsAct1 ) promoter and rice Actin 1 (OsAct1 ) terminator.

Article Title: Streptomyces lividans Blasticidin S Deaminase and Its Application in Engineering a Blasticidin S-Producing Strain for Ease of Genetic Manipulation
Article Snippet: .. PCR was carried out with high-fidelity DNA polymerase (KOD-Plus; Toyobo), and products were gel purified, digested with the appropriate enzymes, and cloned into the corresponding restriction sites of the pET28a(+) vector (Novagen). .. The resulting plasmid, named pWJ4, was used to transform E. coli DH10B for sequencing.

Article Title: Concurrent Overexpression of OsGS1;1 and OsGS2 Genes in Transgenic Rice (Oryza sativa L.): Impact on Tolerance to Abiotic Stresses
Article Snippet: .. The full-length coding sequences of OsGS1;1 (1113 bp) and OsGS2 (1287 bp) were amplified by PCR from rice ( Oryza sativa L. ssp japonica cv Nipponbare) cDNA using a high fidelity DNA polymerase (KOD plus, Toyobo, Japan) and cloned into pCR-4-TOPO vector (Invitrogen, USA). .. Following verification by sequencing, the OsGS1;1 gene was sub-cloned into a Gateway compatible entry vector EV-1 (pL12R34-Ap) in between the rice Actin 2 ( OsAct2 ) promoter and rice Actin 2 ( OsAct2 ) terminator, whereas the OsGS2 gene was cloned into the Gateway compatible EV-2 (pL34R12-Cm-ccdB) vector under the rice Actin 1 ( OsAct1 ) promoter and rice Actin 1 ( OsAct1 ) terminator.

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    Toyobo high fidelity dna polymerase
    Typical mass spectra of four <t>biomarker</t> proteins in E. coli . A) MALDI mass spectra of non-EHEC E. coli strain NBRC12713 and EHEC E. coli strain O157 GTC 14513. Three biomarker peaks, HdeB ( m/z 9066.2 [M+H] + ), ribosomal protein S15 ( m/z 10138.6/10166.6 [M+H] + ) and L25 ( m/z 10676.4/10694.4 [M+H] + ), measured using sinapic acid as the matrix, are shown. B) Biomarker peak of <t>DNA</t> binding protein H-NS ( m/z 15409.4/15425.4 [M+H] + ) for strains O26, O111 and other E. coli .
    High Fidelity Dna Polymerase, supplied by Toyobo, used in various techniques. Bioz Stars score: 93/100, based on 43 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Typical mass spectra of four biomarker proteins in E. coli . A) MALDI mass spectra of non-EHEC E. coli strain NBRC12713 and EHEC E. coli strain O157 GTC 14513. Three biomarker peaks, HdeB ( m/z 9066.2 [M+H] + ), ribosomal protein S15 ( m/z 10138.6/10166.6 [M+H] + ) and L25 ( m/z 10676.4/10694.4 [M+H] + ), measured using sinapic acid as the matrix, are shown. B) Biomarker peak of DNA binding protein H-NS ( m/z 15409.4/15425.4 [M+H] + ) for strains O26, O111 and other E. coli .

    Journal: PLoS ONE

    Article Title: Discrimination of Escherichia coli O157, O26 and O111 from Other Serovars by MALDI-TOF MS Based on the S10-GERMS Method

    doi: 10.1371/journal.pone.0113458

    Figure Lengend Snippet: Typical mass spectra of four biomarker proteins in E. coli . A) MALDI mass spectra of non-EHEC E. coli strain NBRC12713 and EHEC E. coli strain O157 GTC 14513. Three biomarker peaks, HdeB ( m/z 9066.2 [M+H] + ), ribosomal protein S15 ( m/z 10138.6/10166.6 [M+H] + ) and L25 ( m/z 10676.4/10694.4 [M+H] + ), measured using sinapic acid as the matrix, are shown. B) Biomarker peak of DNA binding protein H-NS ( m/z 15409.4/15425.4 [M+H] + ) for strains O26, O111 and other E. coli .

    Article Snippet: In brief, respective regions of ribosomal protein-encoding genes (≈5 kbp) or genes encoding biomarker proteins were amplified using high-fidelity DNA polymerase, KOD plus (Toyobo, Osaka, Japan), and primers designed against the consensus DNA sequences up- and down-stream of the target regions in the E. coli genome sequences in the NCBI database.

    Techniques: Biomarker Assay, Binding Assay

    Determination of the binding sites of CalR3 protein. (A) DNase I footprinting assay of CalR3 on the T-R3L region. The fluorograms correspond to the control reaction without CalR3 protein and to the protection patterns with increasing CalR3 protein (0.12 and 0.24 μg). (B) Nucleotide sequences of the T-R3L promoter region and CalR3-binding site. Solid line, CalR3-binding site; shaded areas, translational start codons; bent arrow, TSPs; boxes, putative –10 and –35 regions. (C) Mutations introduced into the CalR3 binding sites I and II regions. Each probe was 80 bp. Probe m0 was WT DNA containing a 14 bp palindromic sequence in site II. Base substitutions were introduced into probe m0 to produce mutated probes m1, m2, and m1 2, respectively. Altered nucleotides are underlined. (D) EMSAs with WT DNA probe and mutated probes. Each reaction mixture contained 1.5 μM probe and 0.8 μM of purified His 6 -CalR3 protein.

    Journal: Frontiers in Microbiology

    Article Title: A Novel TetR Family Transcriptional Regulator, CalR3, Negatively Controls Calcimycin Biosynthesis in Streptomyces chartreusis NRRL 3882

    doi: 10.3389/fmicb.2017.02371

    Figure Lengend Snippet: Determination of the binding sites of CalR3 protein. (A) DNase I footprinting assay of CalR3 on the T-R3L region. The fluorograms correspond to the control reaction without CalR3 protein and to the protection patterns with increasing CalR3 protein (0.12 and 0.24 μg). (B) Nucleotide sequences of the T-R3L promoter region and CalR3-binding site. Solid line, CalR3-binding site; shaded areas, translational start codons; bent arrow, TSPs; boxes, putative –10 and –35 regions. (C) Mutations introduced into the CalR3 binding sites I and II regions. Each probe was 80 bp. Probe m0 was WT DNA containing a 14 bp palindromic sequence in site II. Base substitutions were introduced into probe m0 to produce mutated probes m1, m2, and m1 2, respectively. Altered nucleotides are underlined. (D) EMSAs with WT DNA probe and mutated probes. Each reaction mixture contained 1.5 μM probe and 0.8 μM of purified His 6 -CalR3 protein.

    Article Snippet: For the complementation of strain GLX26(ΔcalR3 ), the intact calR3 gene was amplified from S. chartreusis NRRL 3882 genomic DNA with the primers calR3-F5 and calR3-F6 using a high-fidelity DNA polymerase (KOD-plus, TOYOBO).

    Techniques: Binding Assay, Footprinting, Sequencing, Purification

    Molecular and biochemical analysis of transgenic rice lines co-overexpressing OsGS1;1 and OsGS2 . (A) PCR amplification of hygromycin phosphotransferase ( hpt ), OsGS1;1 and OSGS2 genes using specific primers in wild type ( wt ), null segregant ( ns ), and five positive T 2 transgenic lines (L1-L5). M: 1Kb DNA ladder (+): positive PCR control (pMDC99) and (–) water blank. (B) Southern blot analysis of wt and five T 2 transgenic lines (L1, L2, L3, L4, and L5), probed with hpt gene probe showing single copy insertion. (C) Semi quantitative RT-PCR showing overexpression of OsGS1;1 and OsGS2 in transgenic lines (L1, L4, and L5) as compared to wt . The rice eEF1α gene was used as a reference gene and rRNA was used as loading control. (D ; top panel) Immunoblot analysis of three transgenic rice lines (L1, L4, and L5) and wt using a recombinant antibody which detects both OsGS1;1 and OsGS2 isoforms (black lines separate spliced regions from same blot) ( D ; bottom panel) Coomassie blue stained Rubisco large subunit (RubL) was used as loading control. (E) Total GS activity of three transgenic rice lines (L1, L4, and L5) in comparison to wt ). One unit of GS activity represents 1.0 μmol of γ-glutamylhydroxamate produced in 20 min. Asterisks above bars indicate significant differences from wt (* at p ≤ 0.05 and ** at p ≤ 0.01).

    Journal: Frontiers in Plant Science

    Article Title: Concurrent Overexpression of OsGS1;1 and OsGS2 Genes in Transgenic Rice (Oryza sativa L.): Impact on Tolerance to Abiotic Stresses

    doi: 10.3389/fpls.2018.00786

    Figure Lengend Snippet: Molecular and biochemical analysis of transgenic rice lines co-overexpressing OsGS1;1 and OsGS2 . (A) PCR amplification of hygromycin phosphotransferase ( hpt ), OsGS1;1 and OSGS2 genes using specific primers in wild type ( wt ), null segregant ( ns ), and five positive T 2 transgenic lines (L1-L5). M: 1Kb DNA ladder (+): positive PCR control (pMDC99) and (–) water blank. (B) Southern blot analysis of wt and five T 2 transgenic lines (L1, L2, L3, L4, and L5), probed with hpt gene probe showing single copy insertion. (C) Semi quantitative RT-PCR showing overexpression of OsGS1;1 and OsGS2 in transgenic lines (L1, L4, and L5) as compared to wt . The rice eEF1α gene was used as a reference gene and rRNA was used as loading control. (D ; top panel) Immunoblot analysis of three transgenic rice lines (L1, L4, and L5) and wt using a recombinant antibody which detects both OsGS1;1 and OsGS2 isoforms (black lines separate spliced regions from same blot) ( D ; bottom panel) Coomassie blue stained Rubisco large subunit (RubL) was used as loading control. (E) Total GS activity of three transgenic rice lines (L1, L4, and L5) in comparison to wt ). One unit of GS activity represents 1.0 μmol of γ-glutamylhydroxamate produced in 20 min. Asterisks above bars indicate significant differences from wt (* at p ≤ 0.05 and ** at p ≤ 0.01).

    Article Snippet: The full-length coding sequences of OsGS1;1 (1113 bp) and OsGS2 (1287 bp) were amplified by PCR from rice ( Oryza sativa L. ssp japonica cv Nipponbare) cDNA using a high fidelity DNA polymerase (KOD plus, Toyobo, Japan) and cloned into pCR-4-TOPO vector (Invitrogen, USA).

    Techniques: Transgenic Assay, Polymerase Chain Reaction, Amplification, Southern Blot, Quantitative RT-PCR, Over Expression, Recombinant, Staining, Activity Assay, Produced

    High-performance liquid chromatography analysis of the metabolite production of S. lividans WJ2. (A) Confirmation of the genotype of the SLBSD mutant WJ2 by PCR with primer Con-BKO (see Table S2 in the supplemental material). M, DNA marker; HXY16, PCR product targeting the SLBSD gene from S. lividans HXY16; WJ2, PCR product targeting the SLBSD gene from S. lividans WJ2. (B) High-performance liquid chromatography comparison of the blasticidin S standard to the purified fermentation broth of S. lividans WJ2 and S. lividans HXY16.

    Journal: Applied and Environmental Microbiology

    Article Title: Streptomyces lividans Blasticidin S Deaminase and Its Application in Engineering a Blasticidin S-Producing Strain for Ease of Genetic Manipulation

    doi: 10.1128/AEM.03254-12

    Figure Lengend Snippet: High-performance liquid chromatography analysis of the metabolite production of S. lividans WJ2. (A) Confirmation of the genotype of the SLBSD mutant WJ2 by PCR with primer Con-BKO (see Table S2 in the supplemental material). M, DNA marker; HXY16, PCR product targeting the SLBSD gene from S. lividans HXY16; WJ2, PCR product targeting the SLBSD gene from S. lividans WJ2. (B) High-performance liquid chromatography comparison of the blasticidin S standard to the purified fermentation broth of S. lividans WJ2 and S. lividans HXY16.

    Article Snippet: PCR was carried out with high-fidelity DNA polymerase (KOD-Plus; Toyobo), and products were gel purified, digested with the appropriate enzymes, and cloned into the corresponding restriction sites of the pET28a(+) vector (Novagen).

    Techniques: High Performance Liquid Chromatography, Mutagenesis, Polymerase Chain Reaction, Marker, Purification

    Assay of the blasticidin S production of the Δ blsF and Δ blsE mutant strains, S. lividans WJ4 and S. lividans WJ5, respectively. (A) Confirmation of the Δ blsF mutant WJ4 by PCR with primer Con-blsF (see Table S2 in the supplemental material). M, DNA marker; WJ2, PCR product targeting blsF from S. lividans WJ2; WJ4, PCR product targeting blsF from S. lividans WJ4. (B) High-performance liquid chromatography comparison of the purified fermentation broth of WJ4 to that of WJ2. (C) Confirmation of the Δ blsE mutant WJ5 by PCR with primer Con-blsE (see Table S2). M, DNA marker; WJ2, PCR product targeting blsE from S. lividans WJ2; WJ5, PCR product targeting blsE from S. lividans WJ5. (D) High-performance liquid chromatography comparison of the untreated fermentation broth of WJ5 to that of WJ2 and HXY16.

    Journal: Applied and Environmental Microbiology

    Article Title: Streptomyces lividans Blasticidin S Deaminase and Its Application in Engineering a Blasticidin S-Producing Strain for Ease of Genetic Manipulation

    doi: 10.1128/AEM.03254-12

    Figure Lengend Snippet: Assay of the blasticidin S production of the Δ blsF and Δ blsE mutant strains, S. lividans WJ4 and S. lividans WJ5, respectively. (A) Confirmation of the Δ blsF mutant WJ4 by PCR with primer Con-blsF (see Table S2 in the supplemental material). M, DNA marker; WJ2, PCR product targeting blsF from S. lividans WJ2; WJ4, PCR product targeting blsF from S. lividans WJ4. (B) High-performance liquid chromatography comparison of the purified fermentation broth of WJ4 to that of WJ2. (C) Confirmation of the Δ blsE mutant WJ5 by PCR with primer Con-blsE (see Table S2). M, DNA marker; WJ2, PCR product targeting blsE from S. lividans WJ2; WJ5, PCR product targeting blsE from S. lividans WJ5. (D) High-performance liquid chromatography comparison of the untreated fermentation broth of WJ5 to that of WJ2 and HXY16.

    Article Snippet: PCR was carried out with high-fidelity DNA polymerase (KOD-Plus; Toyobo), and products were gel purified, digested with the appropriate enzymes, and cloned into the corresponding restriction sites of the pET28a(+) vector (Novagen).

    Techniques: Mutagenesis, Polymerase Chain Reaction, Marker, High Performance Liquid Chromatography, Purification

    Assay of the blasticidin S production of the Δ blsL mutant strain S. lividans WJ3. (A) High-performance liquid chromatography comparison of the blasticidin S standard to the purified fermentation broth of WJ3, WJ2, and HXY16. LC-MS analysis revealed the peak presented in the fermentation broth of LL2 and WJ2, with a dominant peak at m/z 409. (B) MS/MS analysis of the m/z 409 parent ion. Each labeled daughter peak (corresponding structures are shown in the inset) could be derived from demethylblasticidin S. (C) PCR confirmation of the genotype of the Δ blsL mutant WJ3 with primer Con-blsL (see Table S2 in the supplemental material). M, DNA marker; WJ2, PCR product targeting blsL from S. lividans WJ2; WJ3, PCR product targeting blsL from S. lividans WJ3.

    Journal: Applied and Environmental Microbiology

    Article Title: Streptomyces lividans Blasticidin S Deaminase and Its Application in Engineering a Blasticidin S-Producing Strain for Ease of Genetic Manipulation

    doi: 10.1128/AEM.03254-12

    Figure Lengend Snippet: Assay of the blasticidin S production of the Δ blsL mutant strain S. lividans WJ3. (A) High-performance liquid chromatography comparison of the blasticidin S standard to the purified fermentation broth of WJ3, WJ2, and HXY16. LC-MS analysis revealed the peak presented in the fermentation broth of LL2 and WJ2, with a dominant peak at m/z 409. (B) MS/MS analysis of the m/z 409 parent ion. Each labeled daughter peak (corresponding structures are shown in the inset) could be derived from demethylblasticidin S. (C) PCR confirmation of the genotype of the Δ blsL mutant WJ3 with primer Con-blsL (see Table S2 in the supplemental material). M, DNA marker; WJ2, PCR product targeting blsL from S. lividans WJ2; WJ3, PCR product targeting blsL from S. lividans WJ3.

    Article Snippet: PCR was carried out with high-fidelity DNA polymerase (KOD-Plus; Toyobo), and products were gel purified, digested with the appropriate enzymes, and cloned into the corresponding restriction sites of the pET28a(+) vector (Novagen).

    Techniques: Mutagenesis, High Performance Liquid Chromatography, Purification, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Labeling, Derivative Assay, Polymerase Chain Reaction, Marker