e coli dh10b  (Thermo Fisher)


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    Structured Review

    Thermo Fisher e coli dh10b
    mTn5 transposon mutagenesis using an IPTG-controlled conditional suicide plasmid. a Diagram of plasmid pSNC-mTn5. pSNC-mTn5 is a derivative of pMMB-repF that contains a Kan R -tagged mTn5, a lac promoter-controlled hyperactive transposase gene ( tnp H ), and a sacB counter selection marker (with its own promoter). OE and IE are outside and inside ends of the mTn5. b Plasmid and transposon retention frequencies in E. coli <t>DH10B.</t> A “+” symbol for IPTG indicates that the inducer was added to the liquid culture, and a “+” symbol for Suc, Cam, and Kan indicates that the chemicals were added to the plates. Black columns represent plasmid retention frequencies, and the blue column represents Tn5 retention frequency. Results were average of three independent experiments, and bars represent mean ± SD (*p
    E Coli Dh10b, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 68 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    e coli dh10b - by Bioz Stars, 2020-08
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    Images

    1) Product Images from "Efficient transposon mutagenesis mediated by an IPTG-controlled conditional suicide plasmid"

    Article Title: Efficient transposon mutagenesis mediated by an IPTG-controlled conditional suicide plasmid

    Journal: BMC Microbiology

    doi: 10.1186/s12866-018-1319-0

    mTn5 transposon mutagenesis using an IPTG-controlled conditional suicide plasmid. a Diagram of plasmid pSNC-mTn5. pSNC-mTn5 is a derivative of pMMB-repF that contains a Kan R -tagged mTn5, a lac promoter-controlled hyperactive transposase gene ( tnp H ), and a sacB counter selection marker (with its own promoter). OE and IE are outside and inside ends of the mTn5. b Plasmid and transposon retention frequencies in E. coli DH10B. A “+” symbol for IPTG indicates that the inducer was added to the liquid culture, and a “+” symbol for Suc, Cam, and Kan indicates that the chemicals were added to the plates. Black columns represent plasmid retention frequencies, and the blue column represents Tn5 retention frequency. Results were average of three independent experiments, and bars represent mean ± SD (*p
    Figure Legend Snippet: mTn5 transposon mutagenesis using an IPTG-controlled conditional suicide plasmid. a Diagram of plasmid pSNC-mTn5. pSNC-mTn5 is a derivative of pMMB-repF that contains a Kan R -tagged mTn5, a lac promoter-controlled hyperactive transposase gene ( tnp H ), and a sacB counter selection marker (with its own promoter). OE and IE are outside and inside ends of the mTn5. b Plasmid and transposon retention frequencies in E. coli DH10B. A “+” symbol for IPTG indicates that the inducer was added to the liquid culture, and a “+” symbol for Suc, Cam, and Kan indicates that the chemicals were added to the plates. Black columns represent plasmid retention frequencies, and the blue column represents Tn5 retention frequency. Results were average of three independent experiments, and bars represent mean ± SD (*p

    Techniques Used: Mutagenesis, Plasmid Preparation, Selection, Marker, Chick Chorioallantoic Membrane Assay

    Generation of a P. aeruginosa insertion library with pSNC-mTn5ME. a Diagram of pSNC-mTn5ME, a derivative of pSNC-mTn5 that has MEs instead of OE and IE at the termini of mTn5. b Plasmid and transposon retention frequencies in E. coli DH10B. Results were average of three independent experiments, and bars represent mean ± SD (* p
    Figure Legend Snippet: Generation of a P. aeruginosa insertion library with pSNC-mTn5ME. a Diagram of pSNC-mTn5ME, a derivative of pSNC-mTn5 that has MEs instead of OE and IE at the termini of mTn5. b Plasmid and transposon retention frequencies in E. coli DH10B. Results were average of three independent experiments, and bars represent mean ± SD (* p

    Techniques Used: Plasmid Preparation

    IPTG-controlled conditional suicide plasmids. a Plasmid pMMB208 and its conditional-suicide derivatives. pMMB208 contains an RSF1010 oriV for replication and an oriT for conjugation. Genes repA , mobA / repB and repC encode proteins required for plasmid replication, and repF encodes a transcription repressor that binds promoter P4. pMMB208 also has Cam R and lacI Q genes and a P tac promoter. Plasmid pMMB- repF is a derivative of pMMB208 that has a second copy of the repF gene inserted downstream of P tac . Plasmids pMMB- repA K42A and pMMB- repA D139A have a dominant-negative repA mutant gene, either K42A or D139A, inserted downstream of P tac . b Amount of E. coli DH10B cells retaining the indicated plasmids after 24 h growth in the absence of antibiotics, either with or without IPTG induction. Results were average of three independent experiments, and bars represent mean ± SD (standard deviation). * p
    Figure Legend Snippet: IPTG-controlled conditional suicide plasmids. a Plasmid pMMB208 and its conditional-suicide derivatives. pMMB208 contains an RSF1010 oriV for replication and an oriT for conjugation. Genes repA , mobA / repB and repC encode proteins required for plasmid replication, and repF encodes a transcription repressor that binds promoter P4. pMMB208 also has Cam R and lacI Q genes and a P tac promoter. Plasmid pMMB- repF is a derivative of pMMB208 that has a second copy of the repF gene inserted downstream of P tac . Plasmids pMMB- repA K42A and pMMB- repA D139A have a dominant-negative repA mutant gene, either K42A or D139A, inserted downstream of P tac . b Amount of E. coli DH10B cells retaining the indicated plasmids after 24 h growth in the absence of antibiotics, either with or without IPTG induction. Results were average of three independent experiments, and bars represent mean ± SD (standard deviation). * p

    Techniques Used: Plasmid Preparation, Conjugation Assay, Chick Chorioallantoic Membrane Assay, Dominant Negative Mutation, Mutagenesis, Standard Deviation

    2) Product Images from "Following Drug Uptake and Reactions inside Escherichia coli Cells by Raman Microspectroscopy"

    Article Title: Following Drug Uptake and Reactions inside Escherichia coli Cells by Raman Microspectroscopy

    Journal: Biochemistry

    doi: 10.1021/bi500529c

    Partial Raman difference spectra of E. coli DH10B cells producing SHV-1 (top) or KPC-2 (bottom) enzyme after growing in the presence of 10 μg/mL clavulanic acid. * indicates the trans -enamine peak.
    Figure Legend Snippet: Partial Raman difference spectra of E. coli DH10B cells producing SHV-1 (top) or KPC-2 (bottom) enzyme after growing in the presence of 10 μg/mL clavulanic acid. * indicates the trans -enamine peak.

    Techniques Used:

    Quantification of the total amount of SHV-1 or KPC-2 per bacterial cell. (a) Western blot with anti-SHV-1 antibody and anti-DnaK antibody (as a loading control) using lysed E. coli DH10B SHV-1 pBC SK (−) cells and known amounts of pure SHV-1. (b) Western blot with anti-KPC-2 antibody and anti-DnaK antibody (as a loading control) using lysed E. coli DH10B KPC-2 pBC SK (+) cells and known amounts of pure KPC-2.
    Figure Legend Snippet: Quantification of the total amount of SHV-1 or KPC-2 per bacterial cell. (a) Western blot with anti-SHV-1 antibody and anti-DnaK antibody (as a loading control) using lysed E. coli DH10B SHV-1 pBC SK (−) cells and known amounts of pure SHV-1. (b) Western blot with anti-KPC-2 antibody and anti-DnaK antibody (as a loading control) using lysed E. coli DH10B KPC-2 pBC SK (+) cells and known amounts of pure KPC-2.

    Techniques Used: Western Blot

    (a) Partial Raman spectra of (A) freeze-dried E. coli DH10B and (B) freeze-dried E. coli DH10B mixed with 500 μg of clavulanic acid and (C) the Raman difference spectrum (B minus A). (b) Reference curve for freeze-dried mixtures of clavulanic acid with E. coli DH10B cells (plotted as means with n = 3, error bars show ±SD).
    Figure Legend Snippet: (a) Partial Raman spectra of (A) freeze-dried E. coli DH10B and (B) freeze-dried E. coli DH10B mixed with 500 μg of clavulanic acid and (C) the Raman difference spectrum (B minus A). (b) Reference curve for freeze-dried mixtures of clavulanic acid with E. coli DH10B cells (plotted as means with n = 3, error bars show ±SD).

    Techniques Used:

    Partial Raman difference spectrum of E. coli DH10B cells producing SHV-1 grown in the presence of 200 μg/mL tazobactam. The peaks at 1789 and 627 cm –1 represent unreacted tazobactam, and the peak at 1597 cm –1 is assigned to enamine intermediates.
    Figure Legend Snippet: Partial Raman difference spectrum of E. coli DH10B cells producing SHV-1 grown in the presence of 200 μg/mL tazobactam. The peaks at 1789 and 627 cm –1 represent unreacted tazobactam, and the peak at 1597 cm –1 is assigned to enamine intermediates.

    Techniques Used:

    Raman spectra of freeze-dried E. coli DH10B cells producing SHV-1 enzyme in the absence (top) or presence (bottom) of 10 μg/mL clavulanic acid. A, C, G, and T stand for adenine, cytosine, guanine, and thymine, respectively.
    Figure Legend Snippet: Raman spectra of freeze-dried E. coli DH10B cells producing SHV-1 enzyme in the absence (top) or presence (bottom) of 10 μg/mL clavulanic acid. A, C, G, and T stand for adenine, cytosine, guanine, and thymine, respectively.

    Techniques Used:

    Raman difference spectra of E. coli DH10B cells producing (a) SHV-1 E166A, (b) KPC-2 E166A, or (c) no β-lactamase grown in the presence of 10 μg/mL clavulanic acid and (d) cells without β-lactamase grown in the presence of 10 μg/mL mecillinam. * in (a) and (b) denotes the trans -enamine peak. ▼ in (a), (b), and (c) denotes the peak due to intact clavulanic acid.
    Figure Legend Snippet: Raman difference spectra of E. coli DH10B cells producing (a) SHV-1 E166A, (b) KPC-2 E166A, or (c) no β-lactamase grown in the presence of 10 μg/mL clavulanic acid and (d) cells without β-lactamase grown in the presence of 10 μg/mL mecillinam. * in (a) and (b) denotes the trans -enamine peak. ▼ in (a), (b), and (c) denotes the peak due to intact clavulanic acid.

    Techniques Used:

    3) Product Images from "Exploring the Role of a Conserved Class A Residue in the ?-Loop of KPC-2 ?-Lactamase"

    Article Title: Exploring the Role of a Conserved Class A Residue in the ?-Loop of KPC-2 ?-Lactamase

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.348540

    Immunoblot for KPC-2 and, as a loading control, DNA-K, probing E. coli DH10B cells harboring pBR322- catI-bla KPC-2 R164X grown in LB.
    Figure Legend Snippet: Immunoblot for KPC-2 and, as a loading control, DNA-K, probing E. coli DH10B cells harboring pBR322- catI-bla KPC-2 R164X grown in LB.

    Techniques Used:

    4) Product Images from "Chromosome-Encoded Narrow-Spectrum Ambler Class A ?-Lactamase GIL-1 from Citrobacter gillenii ▿"

    Article Title: Chromosome-Encoded Narrow-Spectrum Ambler Class A ?-Lactamase GIL-1 from Citrobacter gillenii ▿

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.01152-06

    Localization of bla GIL-1 in I-CeuI-generated fragments of C. gillenii CIP 106783 T separated by PFGE. Lane A, I-CeuI restriction pattern of E. coli DH10B, used as a control; lane B, I-CeuI restriction pattern of C. gillenii CIP 106783 T ; lane C, hybridization of the I-CeuI restriction pattern of C. gillenii CIP 106783 T with a probe specific for the 16S rRNA gene; lane D, hybridization of the I-CeuI restriction pattern of C. gillenii CIP 106783 T with a probe specific for the bla GIL-1 gene.
    Figure Legend Snippet: Localization of bla GIL-1 in I-CeuI-generated fragments of C. gillenii CIP 106783 T separated by PFGE. Lane A, I-CeuI restriction pattern of E. coli DH10B, used as a control; lane B, I-CeuI restriction pattern of C. gillenii CIP 106783 T ; lane C, hybridization of the I-CeuI restriction pattern of C. gillenii CIP 106783 T with a probe specific for the 16S rRNA gene; lane D, hybridization of the I-CeuI restriction pattern of C. gillenii CIP 106783 T with a probe specific for the bla GIL-1 gene.

    Techniques Used: Generated, Hybridization

    5) Product Images from "Exploring the Role of Residue 228 in Substrate and Inhibitor Recognition by VIM Metallo-β-lactamases"

    Article Title: Exploring the Role of Residue 228 in Substrate and Inhibitor Recognition by VIM Metallo-β-lactamases

    Journal: Biochemistry

    doi: 10.1021/acs.biochem.5b00106

    Characterization of proteins expressed in E. coli DH10B and E. coli BL21(DE3) pLys cells. Steady-state expression of the R228X library in E. coli DH10B was analyzed by immunoblotting, as described in Experimental Procedures. (A) Proteins were detected
    Figure Legend Snippet: Characterization of proteins expressed in E. coli DH10B and E. coli BL21(DE3) pLys cells. Steady-state expression of the R228X library in E. coli DH10B was analyzed by immunoblotting, as described in Experimental Procedures. (A) Proteins were detected

    Techniques Used: Expressing

    6) Product Images from "Structural basis for the recognition of sulfur in phosphorothioated DNA"

    Article Title: Structural basis for the recognition of sulfur in phosphorothioated DNA

    Journal: Nature Communications

    doi: 10.1038/s41467-018-07093-1

    SBD-homologous domains might function to recognize PT-DNA. a Multiple sequence alignment of representative ScoMcrA-SBD homologs from Streptomyces coelicolor (#1), Streptomyces gancidicus (#2), Escherichia coli (#3), and Morganella morganii (#4). The sulfur-recognizing residues are highlighted in yellow. The consensus sequence is shown at the bottom. Right: Domain organizations of these ScoMcrA homologs. b The surface of ScoMcrA-SBD is colored according to conservation scores, which shows that its sulfur-recognizing cavity is highly conserved. c Purified ScoMcrA-SBD domain homologs from Streptomyces gancidicus (#2), Escherichia coli (#3), and Morganella morganii (#4) specifically recognized PT-DNA with the sulfur atom in the R p , but not in the S p , configuration. d Mutation of P482N in the Escherichia coli SBD homolog significantly diminished, while mutation of P607N in the Streptomyces gancidicus SBD homolog disrupted, the association with PT-DNA with the core sequence G PS AAC. e Heterologous expression of scomcrA homologs from S. gancidicus (#2), E. coli (#3), and M. morganii (#4) restricted transfer of the dnd gene cluster from Salmonella enterica , which contains the genes encoding the “writer” proteins of DNA phosphorothioation. Transformation frequencies of empty pBluescript vector (PT − ) and that harboring the dnd gene cluster (PT + ) into E. coli DH10B expressing various scomcrA homologs are shown
    Figure Legend Snippet: SBD-homologous domains might function to recognize PT-DNA. a Multiple sequence alignment of representative ScoMcrA-SBD homologs from Streptomyces coelicolor (#1), Streptomyces gancidicus (#2), Escherichia coli (#3), and Morganella morganii (#4). The sulfur-recognizing residues are highlighted in yellow. The consensus sequence is shown at the bottom. Right: Domain organizations of these ScoMcrA homologs. b The surface of ScoMcrA-SBD is colored according to conservation scores, which shows that its sulfur-recognizing cavity is highly conserved. c Purified ScoMcrA-SBD domain homologs from Streptomyces gancidicus (#2), Escherichia coli (#3), and Morganella morganii (#4) specifically recognized PT-DNA with the sulfur atom in the R p , but not in the S p , configuration. d Mutation of P482N in the Escherichia coli SBD homolog significantly diminished, while mutation of P607N in the Streptomyces gancidicus SBD homolog disrupted, the association with PT-DNA with the core sequence G PS AAC. e Heterologous expression of scomcrA homologs from S. gancidicus (#2), E. coli (#3), and M. morganii (#4) restricted transfer of the dnd gene cluster from Salmonella enterica , which contains the genes encoding the “writer” proteins of DNA phosphorothioation. Transformation frequencies of empty pBluescript vector (PT − ) and that harboring the dnd gene cluster (PT + ) into E. coli DH10B expressing various scomcrA homologs are shown

    Techniques Used: Sequencing, Purification, Mutagenesis, Expressing, Transformation Assay, Plasmid Preparation

    7) Product Images from "Characterization of antimicrobial resistance of Salmonella Newport isolated from animals, the environment, and animal food products in Canada"

    Article Title: Characterization of antimicrobial resistance of Salmonella Newport isolated from animals, the environment, and animal food products in Canada

    Journal:

    doi:

    Plasmid profiles (A) and Southern blot hybridization (B) with a digoxigenin-labelled bla CMY-2 probe of parent and transformant strains of Salmonella Newport. Lane 1, SA00-2987; lane 2, SA00-2987T ( Escherichia coli DH10B transformed with plasmid DNA of
    Figure Legend Snippet: Plasmid profiles (A) and Southern blot hybridization (B) with a digoxigenin-labelled bla CMY-2 probe of parent and transformant strains of Salmonella Newport. Lane 1, SA00-2987; lane 2, SA00-2987T ( Escherichia coli DH10B transformed with plasmid DNA of

    Techniques Used: Plasmid Preparation, Southern Blot, Hybridization, Transformation Assay

    8) Product Images from "Genetic and Biochemical Characterization of the F-ATPase Operon from Streptococcus sanguis 10904"

    Article Title: Genetic and Biochemical Characterization of the F-ATPase Operon from Streptococcus sanguis 10904

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.185.5.1525-1533.2003

    Complementation of an E. coli atpD mutant strain with pDP31 ( E. coli ) and pSSA9 ( S. sanguis ) atpD genes expressed from plasmids. Plasmids and assay conditions are described in Materials and Methods. The extracts were prepared from overnight cultures of each strain. The error bars indicate six repeats with a minimum of two separate extract preparations. The filled squares represent the wild-type E. coli strain DH10B. The filled circles represent the E. coli atpD mutant JP17. The engineered strains are shown in by open diamonds for JP17(pSSA9) and open triangles for JP17(pDP31).
    Figure Legend Snippet: Complementation of an E. coli atpD mutant strain with pDP31 ( E. coli ) and pSSA9 ( S. sanguis ) atpD genes expressed from plasmids. Plasmids and assay conditions are described in Materials and Methods. The extracts were prepared from overnight cultures of each strain. The error bars indicate six repeats with a minimum of two separate extract preparations. The filled squares represent the wild-type E. coli strain DH10B. The filled circles represent the E. coli atpD mutant JP17. The engineered strains are shown in by open diamonds for JP17(pSSA9) and open triangles for JP17(pDP31).

    Techniques Used: Mutagenesis

    9) Product Images from "Structural basis for the recognition of sulfur in phosphorothioated DNA"

    Article Title: Structural basis for the recognition of sulfur in phosphorothioated DNA

    Journal: Nature Communications

    doi: 10.1038/s41467-018-07093-1

    SBD-homologous domains might function to recognize PT-DNA. a Multiple sequence alignment of representative ScoMcrA-SBD homologs from Streptomyces coelicolor (#1), Streptomyces gancidicus (#2), Escherichia coli (#3), and Morganella morganii (#4). The sulfur-recognizing residues are highlighted in yellow. The consensus sequence is shown at the bottom. Right: Domain organizations of these ScoMcrA homologs. b The surface of ScoMcrA-SBD is colored according to conservation scores, which shows that its sulfur-recognizing cavity is highly conserved. c Purified ScoMcrA-SBD domain homologs from Streptomyces gancidicus (#2), Escherichia coli (#3), and Morganella morganii (#4) specifically recognized PT-DNA with the sulfur atom in the R p , but not in the S p , configuration. d Mutation of P482N in the Escherichia coli SBD homolog significantly diminished, while mutation of P607N in the Streptomyces gancidicus SBD homolog disrupted, the association with PT-DNA with the core sequence G PS AAC. e Heterologous expression of scomcrA homologs from S. gancidicus (#2), E. coli (#3), and M. morganii (#4) restricted transfer of the dnd gene cluster from Salmonella enterica , which contains the genes encoding the “writer” proteins of DNA phosphorothioation. Transformation frequencies of empty pBluescript vector (PT − ) and that harboring the dnd gene cluster (PT + ) into E. coli DH10B expressing various scomcrA homologs are shown
    Figure Legend Snippet: SBD-homologous domains might function to recognize PT-DNA. a Multiple sequence alignment of representative ScoMcrA-SBD homologs from Streptomyces coelicolor (#1), Streptomyces gancidicus (#2), Escherichia coli (#3), and Morganella morganii (#4). The sulfur-recognizing residues are highlighted in yellow. The consensus sequence is shown at the bottom. Right: Domain organizations of these ScoMcrA homologs. b The surface of ScoMcrA-SBD is colored according to conservation scores, which shows that its sulfur-recognizing cavity is highly conserved. c Purified ScoMcrA-SBD domain homologs from Streptomyces gancidicus (#2), Escherichia coli (#3), and Morganella morganii (#4) specifically recognized PT-DNA with the sulfur atom in the R p , but not in the S p , configuration. d Mutation of P482N in the Escherichia coli SBD homolog significantly diminished, while mutation of P607N in the Streptomyces gancidicus SBD homolog disrupted, the association with PT-DNA with the core sequence G PS AAC. e Heterologous expression of scomcrA homologs from S. gancidicus (#2), E. coli (#3), and M. morganii (#4) restricted transfer of the dnd gene cluster from Salmonella enterica , which contains the genes encoding the “writer” proteins of DNA phosphorothioation. Transformation frequencies of empty pBluescript vector (PT − ) and that harboring the dnd gene cluster (PT + ) into E. coli DH10B expressing various scomcrA homologs are shown

    Techniques Used: Sequencing, Purification, Mutagenesis, Expressing, Transformation Assay, Plasmid Preparation

    10) Product Images from "Jungle Express is a versatile repressor system for tight transcriptional control"

    Article Title: Jungle Express is a versatile repressor system for tight transcriptional control

    Journal: Nature Communications

    doi: 10.1038/s41467-018-05857-3

    RFP expression from EilR-regulated promoters in E. coli . Single-cell fluorescence of E. coli expressing RFP from EilR-regulated and three common inducible promoters after growth in either defined media with 0.2% glucose ( a ) or LB media ( b ). P JEx promoters were induced with 1 µM CV, P tet with 400 nM anhydrotetracycline, P BAD with 13 mM arabinose and P trc with 1 mM IPTG. c Flow cytometry measurements show the distribution of single-cell fluorescence of stationary phase populations expressing RFP from P JExD after growth without (blue) or with increasing concentrations of CV (purple). The hatched histogram represents autofluorescence of cells lacking rfp . E . coli DH10B was used in a – c , with rfp expressed from medium copy plasmids (p15A ori ). Values and error bars in a and b represent the means and standard deviation of biological triplicate measurements after subtracting background fluorescence emitted by E. coli lacking rfp
    Figure Legend Snippet: RFP expression from EilR-regulated promoters in E. coli . Single-cell fluorescence of E. coli expressing RFP from EilR-regulated and three common inducible promoters after growth in either defined media with 0.2% glucose ( a ) or LB media ( b ). P JEx promoters were induced with 1 µM CV, P tet with 400 nM anhydrotetracycline, P BAD with 13 mM arabinose and P trc with 1 mM IPTG. c Flow cytometry measurements show the distribution of single-cell fluorescence of stationary phase populations expressing RFP from P JExD after growth without (blue) or with increasing concentrations of CV (purple). The hatched histogram represents autofluorescence of cells lacking rfp . E . coli DH10B was used in a – c , with rfp expressed from medium copy plasmids (p15A ori ). Values and error bars in a and b represent the means and standard deviation of biological triplicate measurements after subtracting background fluorescence emitted by E. coli lacking rfp

    Techniques Used: Expressing, Fluorescence, Flow Cytometry, Cytometry, Standard Deviation

    11) Product Images from "Lysine Acetylation Regulates Alanyl-tRNA Synthetase Activity in Escherichia coli"

    Article Title: Lysine Acetylation Regulates Alanyl-tRNA Synthetase Activity in Escherichia coli

    Journal: Genes

    doi: 10.3390/genes9100473

    Expression of two CobB isoforms (CobB-L and CobB-S) in E. coli . ( a ) The primary sequence of N-terminal extension in CobB-L. The single and double underlines show the first methionine (Met) of CobB-L and CobB-S, respectively. The open and gray boxes indicate basic and hydrophobic amino acids, respectively (without Met). ( b ) Schematic representation of cobB -containing fragments in pACYC184P plasmid derivatives. ( c ) Expression of CobB isoforms in E. coli . Ten micrograms of lysate from E. coli DH10B harboring the pACYC184P derivatives was separated by 15% SDS-PAGE and analyzed by Western blotting using an anti-His-tag antibody to detect His-tagged CobB protein. ( d ) The nucleotide sequence of the cobB region. Open and gray boxes show the 3′-region of nagK and 5′-region of the cobB gene, respectively. The promoter containing −10 and −35 (black boxes) for CobB-L and CobB-S was predicted by BPROM of the Softberry web tool.
    Figure Legend Snippet: Expression of two CobB isoforms (CobB-L and CobB-S) in E. coli . ( a ) The primary sequence of N-terminal extension in CobB-L. The single and double underlines show the first methionine (Met) of CobB-L and CobB-S, respectively. The open and gray boxes indicate basic and hydrophobic amino acids, respectively (without Met). ( b ) Schematic representation of cobB -containing fragments in pACYC184P plasmid derivatives. ( c ) Expression of CobB isoforms in E. coli . Ten micrograms of lysate from E. coli DH10B harboring the pACYC184P derivatives was separated by 15% SDS-PAGE and analyzed by Western blotting using an anti-His-tag antibody to detect His-tagged CobB protein. ( d ) The nucleotide sequence of the cobB region. Open and gray boxes show the 3′-region of nagK and 5′-region of the cobB gene, respectively. The promoter containing −10 and −35 (black boxes) for CobB-L and CobB-S was predicted by BPROM of the Softberry web tool.

    Techniques Used: Expressing, Sequencing, Plasmid Preparation, SDS Page, Western Blot

    12) Product Images from "Functional Characterization of IS1999, an IS4 Family Element Involved in Mobilization and Expression of ?-Lactam Resistance Genes"

    Article Title: Functional Characterization of IS1999, an IS4 Family Element Involved in Mobilization and Expression of ?-Lactam Resistance Genes

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.00375-06

    Hybridizations of EcoRV-digested whole-cell DNAs of E. coli DH10B (pA-1) (lane 1), K. pneumoniae 11978 (lane 2), E. coli DH10B (lane 3), and P. aeruginosa 1 (lane 4). (A) bla OXA-48 -specific hybridization. (B) IS 1999 -specific hybridization.
    Figure Legend Snippet: Hybridizations of EcoRV-digested whole-cell DNAs of E. coli DH10B (pA-1) (lane 1), K. pneumoniae 11978 (lane 2), E. coli DH10B (lane 3), and P. aeruginosa 1 (lane 4). (A) bla OXA-48 -specific hybridization. (B) IS 1999 -specific hybridization.

    Techniques Used: Hybridization

    13) Product Images from "Structural basis for the recognition of sulfur in phosphorothioated DNA"

    Article Title: Structural basis for the recognition of sulfur in phosphorothioated DNA

    Journal: Nature Communications

    doi: 10.1038/s41467-018-07093-1

    SBD-homologous domains might function to recognize PT-DNA. a Multiple sequence alignment of representative ScoMcrA-SBD homologs from Streptomyces coelicolor (#1), Streptomyces gancidicus (#2), Escherichia coli (#3), and Morganella morganii (#4). The sulfur-recognizing residues are highlighted in yellow. The consensus sequence is shown at the bottom. Right: Domain organizations of these ScoMcrA homologs. b The surface of ScoMcrA-SBD is colored according to conservation scores, which shows that its sulfur-recognizing cavity is highly conserved. c Purified ScoMcrA-SBD domain homologs from Streptomyces gancidicus (#2), Escherichia coli (#3), and Morganella morganii (#4) specifically recognized PT-DNA with the sulfur atom in the R p , but not in the S p , configuration. d Mutation of P482N in the Escherichia coli SBD homolog significantly diminished, while mutation of P607N in the Streptomyces gancidicus SBD homolog disrupted, the association with PT-DNA with the core sequence G PS AAC. e Heterologous expression of scomcrA homologs from S. gancidicus (#2), E. coli (#3), and M. morganii (#4) restricted transfer of the dnd gene cluster from Salmonella enterica , which contains the genes encoding the “writer” proteins of DNA phosphorothioation. Transformation frequencies of empty pBluescript vector (PT − ) and that harboring the dnd gene cluster (PT + ) into E. coli DH10B expressing various scomcrA homologs are shown
    Figure Legend Snippet: SBD-homologous domains might function to recognize PT-DNA. a Multiple sequence alignment of representative ScoMcrA-SBD homologs from Streptomyces coelicolor (#1), Streptomyces gancidicus (#2), Escherichia coli (#3), and Morganella morganii (#4). The sulfur-recognizing residues are highlighted in yellow. The consensus sequence is shown at the bottom. Right: Domain organizations of these ScoMcrA homologs. b The surface of ScoMcrA-SBD is colored according to conservation scores, which shows that its sulfur-recognizing cavity is highly conserved. c Purified ScoMcrA-SBD domain homologs from Streptomyces gancidicus (#2), Escherichia coli (#3), and Morganella morganii (#4) specifically recognized PT-DNA with the sulfur atom in the R p , but not in the S p , configuration. d Mutation of P482N in the Escherichia coli SBD homolog significantly diminished, while mutation of P607N in the Streptomyces gancidicus SBD homolog disrupted, the association with PT-DNA with the core sequence G PS AAC. e Heterologous expression of scomcrA homologs from S. gancidicus (#2), E. coli (#3), and M. morganii (#4) restricted transfer of the dnd gene cluster from Salmonella enterica , which contains the genes encoding the “writer” proteins of DNA phosphorothioation. Transformation frequencies of empty pBluescript vector (PT − ) and that harboring the dnd gene cluster (PT + ) into E. coli DH10B expressing various scomcrA homologs are shown

    Techniques Used: Sequencing, Purification, Mutagenesis, Expressing, Transformation Assay, Plasmid Preparation

    14) Product Images from "Development of a Sensitive and Specific Enzyme-Linked Immunosorbent Assay for Detecting and Quantifying CMY-2 and SHV ?-Lactamases"

    Article Title: Development of a Sensitive and Specific Enzyme-Linked Immunosorbent Assay for Detecting and Quantifying CMY-2 and SHV ?-Lactamases

    Journal: Journal of Clinical Microbiology

    doi: 10.1128/JCM.40.6.1947-1957.2002

    Immunoblotting. (a) Immunoblot of various amounts of purified CMY-2 β-lactamase probed with 1 μg of anti-CMY-2 antibody/ml. (b) Immunoblot of various amounts of purified SHV-1 β-lactamase probed with 1 μg of anti-SHV-1 antibody/ml. (c) Immunoblot of various β-lactamase-producing strains probed with 1 μg of anti-CMY-2 antibody/ml. Strains, listed from left to right, included E coli DH10B carrying plasmid pBC SK(−) with the SHV-1 β-lactamase, strains producing K-1 and ACT-1 β-lactamases, strain DH5α/pUC18 producing the TEM-1 β-lactamase, a cefepime-resistant E. aerogenes strain producing a β-lactamase (EA), and a strain expressing the P99 Amp C β-lactamase; in addition, E. coli J53-2-derived strains 194-61 and 194 and E. coli strain 20 (EC20) are clinical and laboratory strains producing CMY-2 β-lactamase. (d) Identical immunoblots of strains E. coli DH10B/pUC18 producing TEM-1 β-lactamase, E. coli DH10B/pBC SK(−) producing SHV-1 β-lactamase, and E. coli J53-2-derived 194-61 producing CMY-2 β-lactamase probed with anti-TEM antibody (1:100 dilution) or 1 μg of anti-SHV antibody/ml.
    Figure Legend Snippet: Immunoblotting. (a) Immunoblot of various amounts of purified CMY-2 β-lactamase probed with 1 μg of anti-CMY-2 antibody/ml. (b) Immunoblot of various amounts of purified SHV-1 β-lactamase probed with 1 μg of anti-SHV-1 antibody/ml. (c) Immunoblot of various β-lactamase-producing strains probed with 1 μg of anti-CMY-2 antibody/ml. Strains, listed from left to right, included E coli DH10B carrying plasmid pBC SK(−) with the SHV-1 β-lactamase, strains producing K-1 and ACT-1 β-lactamases, strain DH5α/pUC18 producing the TEM-1 β-lactamase, a cefepime-resistant E. aerogenes strain producing a β-lactamase (EA), and a strain expressing the P99 Amp C β-lactamase; in addition, E. coli J53-2-derived strains 194-61 and 194 and E. coli strain 20 (EC20) are clinical and laboratory strains producing CMY-2 β-lactamase. (d) Identical immunoblots of strains E. coli DH10B/pUC18 producing TEM-1 β-lactamase, E. coli DH10B/pBC SK(−) producing SHV-1 β-lactamase, and E. coli J53-2-derived 194-61 producing CMY-2 β-lactamase probed with anti-TEM antibody (1:100 dilution) or 1 μg of anti-SHV antibody/ml.

    Techniques Used: Purification, Plasmid Preparation, Activated Clotting Time Assay, Transmission Electron Microscopy, Expressing, Derivative Assay, Western Blot

    15) Product Images from "Jungle Express is a versatile repressor system for tight transcriptional control"

    Article Title: Jungle Express is a versatile repressor system for tight transcriptional control

    Journal: Nature Communications

    doi: 10.1038/s41467-018-05857-3

    RFP expression from EilR-regulated promoters in E. coli . Single-cell fluorescence of E. coli expressing RFP from EilR-regulated and three common inducible promoters after growth in either defined media with 0.2% glucose ( a ) or LB media ( b ). P JEx promoters were induced with 1 µM CV, P tet with 400 nM anhydrotetracycline, P BAD with 13 mM arabinose and P trc with 1 mM IPTG. c Flow cytometry measurements show the distribution of single-cell fluorescence of stationary phase populations expressing RFP from P JExD after growth without (blue) or with increasing concentrations of CV (purple). The hatched histogram represents autofluorescence of cells lacking rfp . E . coli DH10B was used in a – c , with rfp expressed from medium copy plasmids (p15A ori ). Values and error bars in a and b represent the means and standard deviation of biological triplicate measurements after subtracting background fluorescence emitted by E. coli lacking rfp
    Figure Legend Snippet: RFP expression from EilR-regulated promoters in E. coli . Single-cell fluorescence of E. coli expressing RFP from EilR-regulated and three common inducible promoters after growth in either defined media with 0.2% glucose ( a ) or LB media ( b ). P JEx promoters were induced with 1 µM CV, P tet with 400 nM anhydrotetracycline, P BAD with 13 mM arabinose and P trc with 1 mM IPTG. c Flow cytometry measurements show the distribution of single-cell fluorescence of stationary phase populations expressing RFP from P JExD after growth without (blue) or with increasing concentrations of CV (purple). The hatched histogram represents autofluorescence of cells lacking rfp . E . coli DH10B was used in a – c , with rfp expressed from medium copy plasmids (p15A ori ). Values and error bars in a and b represent the means and standard deviation of biological triplicate measurements after subtracting background fluorescence emitted by E. coli lacking rfp

    Techniques Used: Expressing, Fluorescence, Flow Cytometry, Cytometry, Standard Deviation

    16) Product Images from "Construction of an Excisable Bacterial Artificial Chromosome Containing a Full-Length Infectious Clone of Herpes Simplex Virus Type 1: Viruses Reconstituted from the Clone Exhibit Wild-Type Properties In Vitro and In Vivo"

    Article Title: Construction of an Excisable Bacterial Artificial Chromosome Containing a Full-Length Infectious Clone of Herpes Simplex Virus Type 1: Viruses Reconstituted from the Clone Exhibit Wild-Type Properties In Vitro and In Vivo

    Journal: Journal of Virology

    doi: 10.1128/JVI.77.2.1382-1391.2003

    Strategy used to clone the entire HSV-1(F) genome into a BAC. Schematic diagrams of the arrangement of the genome of HSV-1(F) and relevant domains of the recombinant viruses and HSV-BACsare shown. Line 1, a linear representation of the HSV-1(F)Δ305 genome. The unique sequences are represented as unique long (U L ) and short (U S ), and the terminal repeats flanking them are shown as open rectangles with their designation given above. Line 2, an expanded section of the domain encoding U L 1 to U L 5. Arrows indicate the polarity and position of each open reading frame. Line 3, an intergenic region between U L 3 and U L 4 open reading frames of HSV-1(F)Δ305. Open reading frames and poly(A) signals are shown as open rectangles and circles, respectively. The Bam HI site used for the insertion of the BAC is also shown. Line 4, an intergenic region between U L 3 and U L 4 of R7205. A chimeric α27-tk gene and upstream poly(A) signal were inserted into the Bam HI site placed between the U L 3 and U L 4 open reading frames. Line 5, sequence of the recombinant virus, YK301, which was selected in the presence of BUdR on 143TK − cells from among the progeny of transfection of rabbit skin cells with intact R7205 DNA and plasmid pB2. The BAC sequence and the poly(A) signals are shown as open rectangles. loxP sites are represented as filled rectangles. Line 6, arrangement of plasmid pYEbac101 maintained in E. coli YEbac101, which was generated by electroporation of circular viral DNA of YK301 into E. coli DH10B. The expansion of the intergenic region between the U L 3 and U L 4 open reading frames of pYEbac101 is also shown. Line 7, sequence of YK302, which was reconstituted by transfection of pYEbac101 into rabbit skin cells. Line 8, arrangement of YK303 selected in the presence of HAT on 143TK − cells from among the progeny of transfection of rabbit skin cells with intact YK302 DNA and plasmid pRB4867 containing the Bam HI Q fragment of HSV-1(F). The expansion of the intergenic region between open reading frames U L 3 and U L 4 of YK303 is also shown. Line 9, arrangement of plasmid pYEbac102 maintained in E. coli YEbac102, constructed by electroporation of circular DNA of YK303 into E. coli DH10B. Line 10, sequence of YK304, reconstituted by transfection of pYEbac102 into rabbit skin cells. Line 11, sequence of the BAC-excised recombinant virus, generated by coinfection of Vero cells with YK304 and a recombinant adenovirus AxCANCre. As a result of Cre-mediated site-specific recombination, the BAC vector backbone was excised and a single loxP site remained in the intergenic region between open reading frames U L 3 and U L 4. Restriction sites: B, Bam HI; Kp, Kpn I; H, Hin dIII.
    Figure Legend Snippet: Strategy used to clone the entire HSV-1(F) genome into a BAC. Schematic diagrams of the arrangement of the genome of HSV-1(F) and relevant domains of the recombinant viruses and HSV-BACsare shown. Line 1, a linear representation of the HSV-1(F)Δ305 genome. The unique sequences are represented as unique long (U L ) and short (U S ), and the terminal repeats flanking them are shown as open rectangles with their designation given above. Line 2, an expanded section of the domain encoding U L 1 to U L 5. Arrows indicate the polarity and position of each open reading frame. Line 3, an intergenic region between U L 3 and U L 4 open reading frames of HSV-1(F)Δ305. Open reading frames and poly(A) signals are shown as open rectangles and circles, respectively. The Bam HI site used for the insertion of the BAC is also shown. Line 4, an intergenic region between U L 3 and U L 4 of R7205. A chimeric α27-tk gene and upstream poly(A) signal were inserted into the Bam HI site placed between the U L 3 and U L 4 open reading frames. Line 5, sequence of the recombinant virus, YK301, which was selected in the presence of BUdR on 143TK − cells from among the progeny of transfection of rabbit skin cells with intact R7205 DNA and plasmid pB2. The BAC sequence and the poly(A) signals are shown as open rectangles. loxP sites are represented as filled rectangles. Line 6, arrangement of plasmid pYEbac101 maintained in E. coli YEbac101, which was generated by electroporation of circular viral DNA of YK301 into E. coli DH10B. The expansion of the intergenic region between the U L 3 and U L 4 open reading frames of pYEbac101 is also shown. Line 7, sequence of YK302, which was reconstituted by transfection of pYEbac101 into rabbit skin cells. Line 8, arrangement of YK303 selected in the presence of HAT on 143TK − cells from among the progeny of transfection of rabbit skin cells with intact YK302 DNA and plasmid pRB4867 containing the Bam HI Q fragment of HSV-1(F). The expansion of the intergenic region between open reading frames U L 3 and U L 4 of YK303 is also shown. Line 9, arrangement of plasmid pYEbac102 maintained in E. coli YEbac102, constructed by electroporation of circular DNA of YK303 into E. coli DH10B. Line 10, sequence of YK304, reconstituted by transfection of pYEbac102 into rabbit skin cells. Line 11, sequence of the BAC-excised recombinant virus, generated by coinfection of Vero cells with YK304 and a recombinant adenovirus AxCANCre. As a result of Cre-mediated site-specific recombination, the BAC vector backbone was excised and a single loxP site remained in the intergenic region between open reading frames U L 3 and U L 4. Restriction sites: B, Bam HI; Kp, Kpn I; H, Hin dIII.

    Techniques Used: BAC Assay, Recombinant, Sequencing, Transfection, Plasmid Preparation, Generated, Electroporation, HAT Assay, Construct

    17) Product Images from "Structural basis for the recognition of sulfur in phosphorothioated DNA"

    Article Title: Structural basis for the recognition of sulfur in phosphorothioated DNA

    Journal: Nature Communications

    doi: 10.1038/s41467-018-07093-1

    SBD-homologous domains might function to recognize PT-DNA. a Multiple sequence alignment of representative ScoMcrA-SBD homologs from Streptomyces coelicolor (#1), Streptomyces gancidicus (#2), Escherichia coli (#3), and Morganella morganii (#4). The sulfur-recognizing residues are highlighted in yellow. The consensus sequence is shown at the bottom. Right: Domain organizations of these ScoMcrA homologs. b The surface of ScoMcrA-SBD is colored according to conservation scores, which shows that its sulfur-recognizing cavity is highly conserved. c Purified ScoMcrA-SBD domain homologs from Streptomyces gancidicus (#2), Escherichia coli (#3), and Morganella morganii (#4) specifically recognized PT-DNA with the sulfur atom in the R p , but not in the S p , configuration. d Mutation of P482N in the Escherichia coli SBD homolog significantly diminished, while mutation of P607N in the Streptomyces gancidicus SBD homolog disrupted, the association with PT-DNA with the core sequence G PS AAC. e Heterologous expression of scomcrA homologs from S. gancidicus (#2), E. coli (#3), and M. morganii (#4) restricted transfer of the dnd gene cluster from Salmonella enterica , which contains the genes encoding the “writer” proteins of DNA phosphorothioation. Transformation frequencies of empty pBluescript vector (PT − ) and that harboring the dnd gene cluster (PT + ) into E. coli DH10B expressing various scomcrA homologs are shown
    Figure Legend Snippet: SBD-homologous domains might function to recognize PT-DNA. a Multiple sequence alignment of representative ScoMcrA-SBD homologs from Streptomyces coelicolor (#1), Streptomyces gancidicus (#2), Escherichia coli (#3), and Morganella morganii (#4). The sulfur-recognizing residues are highlighted in yellow. The consensus sequence is shown at the bottom. Right: Domain organizations of these ScoMcrA homologs. b The surface of ScoMcrA-SBD is colored according to conservation scores, which shows that its sulfur-recognizing cavity is highly conserved. c Purified ScoMcrA-SBD domain homologs from Streptomyces gancidicus (#2), Escherichia coli (#3), and Morganella morganii (#4) specifically recognized PT-DNA with the sulfur atom in the R p , but not in the S p , configuration. d Mutation of P482N in the Escherichia coli SBD homolog significantly diminished, while mutation of P607N in the Streptomyces gancidicus SBD homolog disrupted, the association with PT-DNA with the core sequence G PS AAC. e Heterologous expression of scomcrA homologs from S. gancidicus (#2), E. coli (#3), and M. morganii (#4) restricted transfer of the dnd gene cluster from Salmonella enterica , which contains the genes encoding the “writer” proteins of DNA phosphorothioation. Transformation frequencies of empty pBluescript vector (PT − ) and that harboring the dnd gene cluster (PT + ) into E. coli DH10B expressing various scomcrA homologs are shown

    Techniques Used: Sequencing, Purification, Mutagenesis, Expressing, Transformation Assay, Plasmid Preparation

    18) Product Images from "Genetic Circuit Performance under Conditions Relevant for Industrial Bioreactors"

    Article Title: Genetic Circuit Performance under Conditions Relevant for Industrial Bioreactors

    Journal: ACS synthetic biology

    doi: 10.1021/sb3000832

    Media and Strain Impact on RBS selection The effect of varying the strength of the RBS connecting the arabinose sensor to the AND gate is shown. The Δ G tot ]. A more positive Δ G tot ]. Fitness scores the accuracy and function of the gate in each condition. A higher fitness indicates that the circuit is properly performing the AND function. The fitness was calculated as follows: First, the baseline fluorescence (-/-) was subtracted from each state. Then, the fluorescence of all states were normalized to the highest value of a partially induced state (-/+, +/-). Finally, the partially induced states were subtracted from the ON state (+/+). The RBSs characterized in this manuscript are colored (RBS A : green, RBS B : orange, RBS C : red, and RBS D ]. The calculated fitness is shown for the four RBS's studied for different media and strains. The media are LB broth (diamonds), minimal media containing 5 g/L yeast extract (5Y, triangles), and unsupplemented minimal media (circles). E. coli DH10B was measured in LB and 5Y, and E. coli DS68637 † was measured in unsupplemented minimal medium. Error bars represent 1 standard deviation of three experiments.
    Figure Legend Snippet: Media and Strain Impact on RBS selection The effect of varying the strength of the RBS connecting the arabinose sensor to the AND gate is shown. The Δ G tot ]. A more positive Δ G tot ]. Fitness scores the accuracy and function of the gate in each condition. A higher fitness indicates that the circuit is properly performing the AND function. The fitness was calculated as follows: First, the baseline fluorescence (-/-) was subtracted from each state. Then, the fluorescence of all states were normalized to the highest value of a partially induced state (-/+, +/-). Finally, the partially induced states were subtracted from the ON state (+/+). The RBSs characterized in this manuscript are colored (RBS A : green, RBS B : orange, RBS C : red, and RBS D ]. The calculated fitness is shown for the four RBS's studied for different media and strains. The media are LB broth (diamonds), minimal media containing 5 g/L yeast extract (5Y, triangles), and unsupplemented minimal media (circles). E. coli DH10B was measured in LB and 5Y, and E. coli DS68637 † was measured in unsupplemented minimal medium. Error bars represent 1 standard deviation of three experiments.

    Techniques Used: Selection, Fluorescence, Standard Deviation

    Impact of Media on Gate Performance in E. coli DH10B Media composition affects the performance of the AND (RBS B ) and NOR gates. E. coli DH10B carrying the (A) AND or (B) NOR gate were grown both uninduced (white bars) and induced (black bars) in media of varying composition. The media composition is listed below the data. LB-Miller (LB) contains 10 g/L Tryptone, 5 g/L yeast extract, and 10 g/L NaCl. The other media is either unsupplemented minimal media (-) or minimal media supplemented as follows: 1 g/L Yeast Extract (1Y), 1 g/L Tryptone (1T), 5 g/L yeast extract (5Y), 5 g/L Tryptone (5T), 1 g/L yeast extract + 1 g/L Tryptone (1Y1T), 5 g/L yeast extract + 5 g/L tryptone (5Y5T). The output of the reference plasmid pFM46 (dotted line) is shown for comparison. All cultures were measured after 9 hours, except the cultures that were grown on minimal (unsupplemented) media, which were measured after 24 hours. Induced AND gates never grew on minimal media. The error bars represent the standard deviation of three experiments performed on different days.
    Figure Legend Snippet: Impact of Media on Gate Performance in E. coli DH10B Media composition affects the performance of the AND (RBS B ) and NOR gates. E. coli DH10B carrying the (A) AND or (B) NOR gate were grown both uninduced (white bars) and induced (black bars) in media of varying composition. The media composition is listed below the data. LB-Miller (LB) contains 10 g/L Tryptone, 5 g/L yeast extract, and 10 g/L NaCl. The other media is either unsupplemented minimal media (-) or minimal media supplemented as follows: 1 g/L Yeast Extract (1Y), 1 g/L Tryptone (1T), 5 g/L yeast extract (5Y), 5 g/L Tryptone (5T), 1 g/L yeast extract + 1 g/L Tryptone (1Y1T), 5 g/L yeast extract + 5 g/L tryptone (5Y5T). The output of the reference plasmid pFM46 (dotted line) is shown for comparison. All cultures were measured after 9 hours, except the cultures that were grown on minimal (unsupplemented) media, which were measured after 24 hours. Induced AND gates never grew on minimal media. The error bars represent the standard deviation of three experiments performed on different days.

    Techniques Used: Plasmid Preparation, Standard Deviation

    Comparison of Gate Performance in E. coli DH10B and E. coli DS68637 † Gate performance is measured in DH10B grown on LB and in DS68637 † grown on unsupplemented minimal media (MM). (A) The output of the AND gate (RBS B ) is shown for four combinations of inputs: -/- (no inducers), +/- (1.3 mM arabinose), -/+ (0.63 mM salicylate), +/+ (both inducers). The output of the reference plasmid pFM46 (S, black bars) is shown for both strain/media combinations. (B) The output of the NOR gate is shown for four combinations of inputs: -/- (no inducers), +/- (1.3 mM arabinose), -/+ (100 ng/ml aTc), +/+ (both inducers). In E. coli ]. In DS68637 † , the AND and NOR gates were both measured after 24 hours due to slower growth on the minimal media. The error bars represent the standard deviation of three experiments performed on different days.
    Figure Legend Snippet: Comparison of Gate Performance in E. coli DH10B and E. coli DS68637 † Gate performance is measured in DH10B grown on LB and in DS68637 † grown on unsupplemented minimal media (MM). (A) The output of the AND gate (RBS B ) is shown for four combinations of inputs: -/- (no inducers), +/- (1.3 mM arabinose), -/+ (0.63 mM salicylate), +/+ (both inducers). The output of the reference plasmid pFM46 (S, black bars) is shown for both strain/media combinations. (B) The output of the NOR gate is shown for four combinations of inputs: -/- (no inducers), +/- (1.3 mM arabinose), -/+ (100 ng/ml aTc), +/+ (both inducers). In E. coli ]. In DS68637 † , the AND and NOR gates were both measured after 24 hours due to slower growth on the minimal media. The error bars represent the standard deviation of three experiments performed on different days.

    Techniques Used: Plasmid Preparation, Standard Deviation

    Performance of an AND gate and Reference Plasmid in a Microreactor and 10 L Bioreactor Cultures of E. coli DS68637 carrying the RBS B AND gate and a reference plasmid (pFM46) were grown in a BioLector microreactor on a 1 ml batch of rich 2xYT medium. (A) ). The reference plasmid data is shown as a dashed line. (B) The performance of E. coli DH10B carrying the RBS B AND gate is shown in a 10 L bioreactor. Both inducers (1.30 mM arabinose and 0.63 mM salicylate) are added at 42.5 hours. The fluorescence per dry cell weight (DCW) is shown for three fermentations in which the amount of yeast extract in the feed is varied: 0 g/kg (squares), 20 g/kg (diamonds), and 100 g/kg (circles). The glucose feed is initiated at 15 hours after inoculation and is exponentially increased over the course of the fermentation.
    Figure Legend Snippet: Performance of an AND gate and Reference Plasmid in a Microreactor and 10 L Bioreactor Cultures of E. coli DS68637 carrying the RBS B AND gate and a reference plasmid (pFM46) were grown in a BioLector microreactor on a 1 ml batch of rich 2xYT medium. (A) ). The reference plasmid data is shown as a dashed line. (B) The performance of E. coli DH10B carrying the RBS B AND gate is shown in a 10 L bioreactor. Both inducers (1.30 mM arabinose and 0.63 mM salicylate) are added at 42.5 hours. The fluorescence per dry cell weight (DCW) is shown for three fermentations in which the amount of yeast extract in the feed is varied: 0 g/kg (squares), 20 g/kg (diamonds), and 100 g/kg (circles). The glucose feed is initiated at 15 hours after inoculation and is exponentially increased over the course of the fermentation.

    Techniques Used: Plasmid Preparation, Fluorescence

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    Article Snippet: .. Lysogeny broth (LB) minimal inhibitory concentration (MICs) determinations for β-lactams were performed on single clones of E. coli DH10B (Invitrogen) harboring pBR322- catI-bla KPC-2 or a variant. ..

    Plasmid Preparation:

    Article Title: Characterization of antimicrobial resistance of Salmonella Newport isolated from animals, the environment, and animal food products in Canada
    Article Snippet: .. The prepared plasmid DNA was used to transform E. coli DH10B (Gibco BRL, Burlington, Ontario) by electroporation ( ) to determine if plasmids that could not be transferred by conjugation, and were therefore likely to not be self-transmissible, encoded drugresistance determinants. ..

    Article Title: Structural basis for the recognition of sulfur in phosphorothioated DNA
    Article Snippet: .. The pACYCDuet™-1 vector and its derivatives carrying FL E. coli , M. morganii , and S. gancidicus ScoMcrA homologs with E. coli promoters (pJTU1673, pJTU1674, and pJTU1675) were introduced to E. coli DH10B, and competent cells of the resulting strains were prepared using the standard calcium chloride protocol. .. Transformation frequency was determined by introducing 100 ng pBluescript SK+ (PT− ) or pJTU1238 (PT+ ) plasmid DNA, which carries the dnd gene cluster from Salmonella enterica serovar Cerro 87 , to the competent cells.

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    Thermo Fisher e coli dh10b
    SBD-homologous domains might function to recognize PT-DNA. a Multiple sequence alignment of representative ScoMcrA-SBD homologs from Streptomyces coelicolor (#1), Streptomyces gancidicus (#2), Escherichia coli (#3), and Morganella morganii (#4). The sulfur-recognizing residues are highlighted in yellow. The consensus sequence is shown at the bottom. Right: Domain organizations of these ScoMcrA homologs. b The surface of ScoMcrA-SBD is colored according to conservation scores, which shows that its sulfur-recognizing cavity is highly conserved. c Purified ScoMcrA-SBD domain homologs from Streptomyces gancidicus (#2), Escherichia coli (#3), and Morganella morganii (#4) specifically recognized PT-DNA with the sulfur atom in the R p , but not in the S p , configuration. d Mutation of P482N in the Escherichia coli SBD homolog significantly diminished, while mutation of P607N in the Streptomyces gancidicus SBD homolog disrupted, the association with PT-DNA with the core sequence G PS AAC. e Heterologous expression of scomcrA homologs from S. gancidicus (#2), E. coli (#3), and M. morganii (#4) restricted transfer of the dnd gene cluster from Salmonella enterica , which contains the genes encoding the “writer” proteins of DNA phosphorothioation. Transformation frequencies of empty pBluescript vector (PT − ) and that harboring the dnd gene cluster (PT + ) into E. coli <t>DH10B</t> expressing various scomcrA homologs are shown
    E Coli Dh10b, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 68 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    SBD-homologous domains might function to recognize PT-DNA. a Multiple sequence alignment of representative ScoMcrA-SBD homologs from Streptomyces coelicolor (#1), Streptomyces gancidicus (#2), Escherichia coli (#3), and Morganella morganii (#4). The sulfur-recognizing residues are highlighted in yellow. The consensus sequence is shown at the bottom. Right: Domain organizations of these ScoMcrA homologs. b The surface of ScoMcrA-SBD is colored according to conservation scores, which shows that its sulfur-recognizing cavity is highly conserved. c Purified ScoMcrA-SBD domain homologs from Streptomyces gancidicus (#2), Escherichia coli (#3), and Morganella morganii (#4) specifically recognized PT-DNA with the sulfur atom in the R p , but not in the S p , configuration. d Mutation of P482N in the Escherichia coli SBD homolog significantly diminished, while mutation of P607N in the Streptomyces gancidicus SBD homolog disrupted, the association with PT-DNA with the core sequence G PS AAC. e Heterologous expression of scomcrA homologs from S. gancidicus (#2), E. coli (#3), and M. morganii (#4) restricted transfer of the dnd gene cluster from Salmonella enterica , which contains the genes encoding the “writer” proteins of DNA phosphorothioation. Transformation frequencies of empty pBluescript vector (PT − ) and that harboring the dnd gene cluster (PT + ) into E. coli DH10B expressing various scomcrA homologs are shown

    Journal: Nature Communications

    Article Title: Structural basis for the recognition of sulfur in phosphorothioated DNA

    doi: 10.1038/s41467-018-07093-1

    Figure Lengend Snippet: SBD-homologous domains might function to recognize PT-DNA. a Multiple sequence alignment of representative ScoMcrA-SBD homologs from Streptomyces coelicolor (#1), Streptomyces gancidicus (#2), Escherichia coli (#3), and Morganella morganii (#4). The sulfur-recognizing residues are highlighted in yellow. The consensus sequence is shown at the bottom. Right: Domain organizations of these ScoMcrA homologs. b The surface of ScoMcrA-SBD is colored according to conservation scores, which shows that its sulfur-recognizing cavity is highly conserved. c Purified ScoMcrA-SBD domain homologs from Streptomyces gancidicus (#2), Escherichia coli (#3), and Morganella morganii (#4) specifically recognized PT-DNA with the sulfur atom in the R p , but not in the S p , configuration. d Mutation of P482N in the Escherichia coli SBD homolog significantly diminished, while mutation of P607N in the Streptomyces gancidicus SBD homolog disrupted, the association with PT-DNA with the core sequence G PS AAC. e Heterologous expression of scomcrA homologs from S. gancidicus (#2), E. coli (#3), and M. morganii (#4) restricted transfer of the dnd gene cluster from Salmonella enterica , which contains the genes encoding the “writer” proteins of DNA phosphorothioation. Transformation frequencies of empty pBluescript vector (PT − ) and that harboring the dnd gene cluster (PT + ) into E. coli DH10B expressing various scomcrA homologs are shown

    Article Snippet: The pACYCDuet™-1 vector and its derivatives carrying FL E. coli , M. morganii , and S. gancidicus ScoMcrA homologs with E. coli promoters (pJTU1673, pJTU1674, and pJTU1675) were introduced to E. coli DH10B, and competent cells of the resulting strains were prepared using the standard calcium chloride protocol.

    Techniques: Sequencing, Purification, Mutagenesis, Expressing, Transformation Assay, Plasmid Preparation

    SBD-homologous domains might function to recognize PT-DNA. a Multiple sequence alignment of representative ScoMcrA-SBD homologs from Streptomyces coelicolor (#1), Streptomyces gancidicus (#2), Escherichia coli (#3), and Morganella morganii (#4). The sulfur-recognizing residues are highlighted in yellow. The consensus sequence is shown at the bottom. Right: Domain organizations of these ScoMcrA homologs. b The surface of ScoMcrA-SBD is colored according to conservation scores, which shows that its sulfur-recognizing cavity is highly conserved. c Purified ScoMcrA-SBD domain homologs from Streptomyces gancidicus (#2), Escherichia coli (#3), and Morganella morganii (#4) specifically recognized PT-DNA with the sulfur atom in the R p , but not in the S p , configuration. d Mutation of P482N in the Escherichia coli SBD homolog significantly diminished, while mutation of P607N in the Streptomyces gancidicus SBD homolog disrupted, the association with PT-DNA with the core sequence G PS AAC. e Heterologous expression of scomcrA homologs from S. gancidicus (#2), E. coli (#3), and M. morganii (#4) restricted transfer of the dnd gene cluster from Salmonella enterica , which contains the genes encoding the “writer” proteins of DNA phosphorothioation. Transformation frequencies of empty pBluescript vector (PT − ) and that harboring the dnd gene cluster (PT + ) into E. coli DH10B expressing various scomcrA homologs are shown

    Journal: Nature Communications

    Article Title: Structural basis for the recognition of sulfur in phosphorothioated DNA

    doi: 10.1038/s41467-018-07093-1

    Figure Lengend Snippet: SBD-homologous domains might function to recognize PT-DNA. a Multiple sequence alignment of representative ScoMcrA-SBD homologs from Streptomyces coelicolor (#1), Streptomyces gancidicus (#2), Escherichia coli (#3), and Morganella morganii (#4). The sulfur-recognizing residues are highlighted in yellow. The consensus sequence is shown at the bottom. Right: Domain organizations of these ScoMcrA homologs. b The surface of ScoMcrA-SBD is colored according to conservation scores, which shows that its sulfur-recognizing cavity is highly conserved. c Purified ScoMcrA-SBD domain homologs from Streptomyces gancidicus (#2), Escherichia coli (#3), and Morganella morganii (#4) specifically recognized PT-DNA with the sulfur atom in the R p , but not in the S p , configuration. d Mutation of P482N in the Escherichia coli SBD homolog significantly diminished, while mutation of P607N in the Streptomyces gancidicus SBD homolog disrupted, the association with PT-DNA with the core sequence G PS AAC. e Heterologous expression of scomcrA homologs from S. gancidicus (#2), E. coli (#3), and M. morganii (#4) restricted transfer of the dnd gene cluster from Salmonella enterica , which contains the genes encoding the “writer” proteins of DNA phosphorothioation. Transformation frequencies of empty pBluescript vector (PT − ) and that harboring the dnd gene cluster (PT + ) into E. coli DH10B expressing various scomcrA homologs are shown

    Article Snippet: E. coli DH10B (Thermo Fisher) and Escherichiacoli BL21(DE3) (Novagen) was grown in Luria Broth medium supplemented with 100 mg/mL ampicillin or 34 mg/mL chloramphenicol as required.

    Techniques: Sequencing, Purification, Mutagenesis, Expressing, Transformation Assay, Plasmid Preparation