e coli top10  (Thermo Fisher)


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    Structured Review

    Thermo Fisher e coli top10
    RT-PCR analysis of tet35 expression. (A) RT-PCR of total RNA from E. coli <t>TOP10</t> cells carrying pATJ1. Lanes: 1, 1-kb ladder; 2, 1.1-kb amplicon obtained with tet35F/R primers; 3, 900-base amplicon obtained with txrF/R primers; 4, positive control using 16S rRNA-specific primers, amplifying a 1.3-kb amplicon; 5, negative control (no RT). (B) RT-PCR of total DNA from E. coli TOP10 cells carrying pJKM115. Lanes 1, 1-kb ladder; 2, 1.1-kb PCR amplicon obtained with tet35F/R primers; 3, no detectable amplicon obtained with txrF/R primers; 4; positive control using 16S rRNA specific primers, amplifying a 1.3-kb amplicon; 5, negative control (no RT).
    E Coli Top10, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 232 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 94 stars, based on 232 article reviews
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    e coli top10 - by Bioz Stars, 2020-08
    94/100 stars

    Images

    1) Product Images from "Genetic Determinants of Tetracycline Resistance in Vibrio harveyi"

    Article Title: Genetic Determinants of Tetracycline Resistance in Vibrio harveyi

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.46.4.1038-1045.2002

    RT-PCR analysis of tet35 expression. (A) RT-PCR of total RNA from E. coli TOP10 cells carrying pATJ1. Lanes: 1, 1-kb ladder; 2, 1.1-kb amplicon obtained with tet35F/R primers; 3, 900-base amplicon obtained with txrF/R primers; 4, positive control using 16S rRNA-specific primers, amplifying a 1.3-kb amplicon; 5, negative control (no RT). (B) RT-PCR of total DNA from E. coli TOP10 cells carrying pJKM115. Lanes 1, 1-kb ladder; 2, 1.1-kb PCR amplicon obtained with tet35F/R primers; 3, no detectable amplicon obtained with txrF/R primers; 4; positive control using 16S rRNA specific primers, amplifying a 1.3-kb amplicon; 5, negative control (no RT).
    Figure Legend Snippet: RT-PCR analysis of tet35 expression. (A) RT-PCR of total RNA from E. coli TOP10 cells carrying pATJ1. Lanes: 1, 1-kb ladder; 2, 1.1-kb amplicon obtained with tet35F/R primers; 3, 900-base amplicon obtained with txrF/R primers; 4, positive control using 16S rRNA-specific primers, amplifying a 1.3-kb amplicon; 5, negative control (no RT). (B) RT-PCR of total DNA from E. coli TOP10 cells carrying pJKM115. Lanes 1, 1-kb ladder; 2, 1.1-kb PCR amplicon obtained with tet35F/R primers; 3, no detectable amplicon obtained with txrF/R primers; 4; positive control using 16S rRNA specific primers, amplifying a 1.3-kb amplicon; 5, negative control (no RT).

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Amplification, Positive Control, Negative Control, Polymerase Chain Reaction

    Accumulation of tetracycline by susceptible E. coli TOP10 cells and resistant clones. [ 3 H]tetracycline was added to cells at time zero. (A) Accumulation in E. coli carrying no plasmid (▪), pATJ1 (•), or pCT71 (▴); (B) effect of CCCP (0.2 mM) on accumulation in E. coli with no plasmid (▪) or pATJ1 (•). The graphs are typical of at least two independent experiments.
    Figure Legend Snippet: Accumulation of tetracycline by susceptible E. coli TOP10 cells and resistant clones. [ 3 H]tetracycline was added to cells at time zero. (A) Accumulation in E. coli carrying no plasmid (▪), pATJ1 (•), or pCT71 (▴); (B) effect of CCCP (0.2 mM) on accumulation in E. coli with no plasmid (▪) or pATJ1 (•). The graphs are typical of at least two independent experiments.

    Techniques Used: Clone Assay, Plasmid Preparation

    2) Product Images from "Buwchitin: A Ruminal Peptide with Antimicrobial Potential against Enterococcus faecalis"

    Article Title: Buwchitin: A Ruminal Peptide with Antimicrobial Potential against Enterococcus faecalis

    Journal: Frontiers in Chemistry

    doi: 10.3389/fchem.2017.00051

    SDS-PAGE analysis of purification steps of buwchitin protein expressed in E. coli TOP10 cells on a 20% denaturing polyacrylamide gel (4 h after induction with 1 mM IPTG). Lane 1, protein molecular weight marker; Lane 2, cell lysate; Lane 3, supernatant; Lane 4, Wash step; Lane 5, eluted buwchitin protein. The arrow indicates band of purified protein of interest. Expected size is 8.35 (±3–4 kDa from His-tag).
    Figure Legend Snippet: SDS-PAGE analysis of purification steps of buwchitin protein expressed in E. coli TOP10 cells on a 20% denaturing polyacrylamide gel (4 h after induction with 1 mM IPTG). Lane 1, protein molecular weight marker; Lane 2, cell lysate; Lane 3, supernatant; Lane 4, Wash step; Lane 5, eluted buwchitin protein. The arrow indicates band of purified protein of interest. Expected size is 8.35 (±3–4 kDa from His-tag).

    Techniques Used: SDS Page, Purification, Molecular Weight, Marker

    3) Product Images from "Quality control of overexpressed membrane proteins"

    Article Title: Quality control of overexpressed membrane proteins

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.0802190105

    Electrophoretic mobility of membrane proteins fused or not fused to GFP. All proteins and GFP fusion proteins were expressed in E. coli MC1061, except for GltP and DctA, which were expressed in E. coli TOP10. Whole-cell samples of cultures induced with
    Figure Legend Snippet: Electrophoretic mobility of membrane proteins fused or not fused to GFP. All proteins and GFP fusion proteins were expressed in E. coli MC1061, except for GltP and DctA, which were expressed in E. coli TOP10. Whole-cell samples of cultures induced with

    Techniques Used:

    4) Product Images from "A Plasmid-Borne System To Assess the Excision and Integration of Staphylococcal Cassette Chromosome mec Mediated by CcrA and CcrB"

    Article Title: A Plasmid-Borne System To Assess the Excision and Integration of Staphylococcal Cassette Chromosome mec Mediated by CcrA and CcrB

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.00078-15

    (A) Detection of recombination after plasmid digestion. Lane 1, 1-kb DNA ladder; lane 2, pWA340; lane 3, pWA341; lane 4, pWA479; lane 5, pWA480. All of the plasmids were extracted from E. coli TOP10. The plasmids in lanes 2 and 3 are the model plasmids for excision (pWA340) and integration (pWA341). The plasmids in lanes 4 and 5 are the model plasmids following the addition of ccrAB and cleavage with BamHI, releasing the 3.3-kb ccrAB genes and leaving the plasmid backbone. Recombination frequency was determined by the difference in intensity between the 9.1-kb intact plasmid band and the 6-kb fragment produced when recombination released the tetracycline resistance gene. A complete description of the plasmids is available in Table S1 in the supplemental material. (B) Recombination frequency monitored throughout 8 to 20 h of E. coli growth and analyzed for excision (pWA479) and integration (pWA480). The value at each time point represents the mean from four separate experiments, with bars showing the standard deviation.
    Figure Legend Snippet: (A) Detection of recombination after plasmid digestion. Lane 1, 1-kb DNA ladder; lane 2, pWA340; lane 3, pWA341; lane 4, pWA479; lane 5, pWA480. All of the plasmids were extracted from E. coli TOP10. The plasmids in lanes 2 and 3 are the model plasmids for excision (pWA340) and integration (pWA341). The plasmids in lanes 4 and 5 are the model plasmids following the addition of ccrAB and cleavage with BamHI, releasing the 3.3-kb ccrAB genes and leaving the plasmid backbone. Recombination frequency was determined by the difference in intensity between the 9.1-kb intact plasmid band and the 6-kb fragment produced when recombination released the tetracycline resistance gene. A complete description of the plasmids is available in Table S1 in the supplemental material. (B) Recombination frequency monitored throughout 8 to 20 h of E. coli growth and analyzed for excision (pWA479) and integration (pWA480). The value at each time point represents the mean from four separate experiments, with bars showing the standard deviation.

    Techniques Used: Plasmid Preparation, Produced, Standard Deviation

    (A) Excision and integration catalyzed by CcrAB or CcrC (WBG8318) from strains carrying SCC mec types II, IV, and V in E. coli TOP10 cells. The ccrAB or ccrC genes were from a series of MRSA or MRSE strains which are described in Table S1 in the supplemental material. Comparison of the amino acid sequences of the CcrAB proteins from the S. aureus strains described here can be found in Fig. S1 in the supplemental material. (B) Excision and integration catalyzed by CcrAB from N315 and MW2 with a change of aa 385 in CcrB. At least three repeats were done for each set of CcrA and CcrB proteins. *, P
    Figure Legend Snippet: (A) Excision and integration catalyzed by CcrAB or CcrC (WBG8318) from strains carrying SCC mec types II, IV, and V in E. coli TOP10 cells. The ccrAB or ccrC genes were from a series of MRSA or MRSE strains which are described in Table S1 in the supplemental material. Comparison of the amino acid sequences of the CcrAB proteins from the S. aureus strains described here can be found in Fig. S1 in the supplemental material. (B) Excision and integration catalyzed by CcrAB from N315 and MW2 with a change of aa 385 in CcrB. At least three repeats were done for each set of CcrA and CcrB proteins. *, P

    Techniques Used:

    5) Product Images from "Transfer of KPC-2 Carbapenemase from Klebsiella pneumoniae to Escherichia coli in a Patient: First Case in Europe ▿"

    Article Title: Transfer of KPC-2 Carbapenemase from Klebsiella pneumoniae to Escherichia coli in a Patient: First Case in Europe ▿

    Journal: Journal of Clinical Microbiology

    doi: 10.1128/JCM.00133-11

    Plasmid analysis of KPC-2-positive K. pneumoniae and E. coli isolates. Plasmids from clinical strains were purified and transformed into E. coli Top10 cells. After plasmid purification of the transformed KPC-2-positive colonies, plasmids were loaded onto 1% agarose gel before (nondigested, ND lanes) or after (digested, D lanes) digestion with EcoRI and HindIII. K stands for K. pneumoniae , and E stands for E. coli . M is a molecular size marker of 75 to 20,000 bp (GeneRuler 1 kb Plus DNA Ladder; Fermentas).
    Figure Legend Snippet: Plasmid analysis of KPC-2-positive K. pneumoniae and E. coli isolates. Plasmids from clinical strains were purified and transformed into E. coli Top10 cells. After plasmid purification of the transformed KPC-2-positive colonies, plasmids were loaded onto 1% agarose gel before (nondigested, ND lanes) or after (digested, D lanes) digestion with EcoRI and HindIII. K stands for K. pneumoniae , and E stands for E. coli . M is a molecular size marker of 75 to 20,000 bp (GeneRuler 1 kb Plus DNA Ladder; Fermentas).

    Techniques Used: Plasmid Preparation, Purification, Transformation Assay, Agarose Gel Electrophoresis, Marker

    6) Product Images from "A New Family of Secreted Toxins in Pathogenic Neisseria Species"

    Article Title: A New Family of Secreted Toxins in Pathogenic Neisseria Species

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1004592

    MafB MGI-1NEM8013 is a bacterial EndoU nuclease. A) Partial sequence alignment of EndoU nuclease domain of Nidovirus Nsp15 protein (NendoU; NP_740619), Xenopus laevis XendoU (Q8JFY9), human placental protein PP11 (HendoU P21128) and MafB MGI-1 from NEM8013 strain (C9X2Z7). The arrows indicate two conserved histidine residues, which are part of the catalytic site of previously characterized EndoU nucleases. Multiple alignment was performed using MUSCLE and shaded using the BoxShade server. Residues that are identical or similar in at least three of the four sequences are shaded with black or grey background respectively. B) Analysis of in vivo impact of MafB MGI-1NEM8013 expression in E. coli . Total RNA from E. coli expressing MafB MGI-1NEM8013 (pBAD33- mafB MGI-1NEM8013 ) was isolated before induction (T0) and 30 min after addition of L-arabinose (T30). Samples were run on 5% denaturing polyacrylamide gels and stained with ethidium bromide. Positions of 23S, 16S, and 5S rRNAs and tRNAs are shown. -, empty vector control; B, E. coli expressing MafB MGI-1NEM8013 from pBAD33 C) RNase activity of purified recombinant MafB MGI-1NEM8013 was assessed by incubating MafB MGI-1NEM8013 -His 6 alone or with MafI MGI-1NEM8013 -His 6 with total RNA isolated from different sources ( N. meningitidis NEM8013, E. coli TOP10 and human epithelial cells FaDu). Each reaction was performed for 30 min at 37°C with 4 µg of RNA in Tris-EDTA buffer. D) Synthetic mRNA of 43 bp was incubated with purified MafB MGI-1NEM8013 -His 6 alone or with MafI MGI-1NEM8013 -His 6 in Tris-EDTA buffer for 15 min at 37°C. The cleavage products were separated by electrophoresis in a 14% polyacrylamide/8M urea gel and were visualized by ethidium bromide staining. E) Synthetic oligoribonucleotides containing several uridylates (U) or none (0) were incubated with purified MafB MGI-1NEM8013 -His 6 in Tris-EDTA buffer for 20 min at 37°C. The reaction products were analyzed by 14% polyacrylamide/8 M urea gel. Sequences of synthetic oligonucleotides used in this experiment are shown.
    Figure Legend Snippet: MafB MGI-1NEM8013 is a bacterial EndoU nuclease. A) Partial sequence alignment of EndoU nuclease domain of Nidovirus Nsp15 protein (NendoU; NP_740619), Xenopus laevis XendoU (Q8JFY9), human placental protein PP11 (HendoU P21128) and MafB MGI-1 from NEM8013 strain (C9X2Z7). The arrows indicate two conserved histidine residues, which are part of the catalytic site of previously characterized EndoU nucleases. Multiple alignment was performed using MUSCLE and shaded using the BoxShade server. Residues that are identical or similar in at least three of the four sequences are shaded with black or grey background respectively. B) Analysis of in vivo impact of MafB MGI-1NEM8013 expression in E. coli . Total RNA from E. coli expressing MafB MGI-1NEM8013 (pBAD33- mafB MGI-1NEM8013 ) was isolated before induction (T0) and 30 min after addition of L-arabinose (T30). Samples were run on 5% denaturing polyacrylamide gels and stained with ethidium bromide. Positions of 23S, 16S, and 5S rRNAs and tRNAs are shown. -, empty vector control; B, E. coli expressing MafB MGI-1NEM8013 from pBAD33 C) RNase activity of purified recombinant MafB MGI-1NEM8013 was assessed by incubating MafB MGI-1NEM8013 -His 6 alone or with MafI MGI-1NEM8013 -His 6 with total RNA isolated from different sources ( N. meningitidis NEM8013, E. coli TOP10 and human epithelial cells FaDu). Each reaction was performed for 30 min at 37°C with 4 µg of RNA in Tris-EDTA buffer. D) Synthetic mRNA of 43 bp was incubated with purified MafB MGI-1NEM8013 -His 6 alone or with MafI MGI-1NEM8013 -His 6 in Tris-EDTA buffer for 15 min at 37°C. The cleavage products were separated by electrophoresis in a 14% polyacrylamide/8M urea gel and were visualized by ethidium bromide staining. E) Synthetic oligoribonucleotides containing several uridylates (U) or none (0) were incubated with purified MafB MGI-1NEM8013 -His 6 in Tris-EDTA buffer for 20 min at 37°C. The reaction products were analyzed by 14% polyacrylamide/8 M urea gel. Sequences of synthetic oligonucleotides used in this experiment are shown.

    Techniques Used: Sequencing, In Vivo, Expressing, Isolation, Staining, Plasmid Preparation, Activity Assay, Purification, Recombinant, Incubation, Electrophoresis

    7) Product Images from "Novel Bacterial Surface Display Systems Based on Outer Membrane Anchoring Elements from the Marine Bacterium Vibrio anguillarum ▿ ▿ †"

    Article Title: Novel Bacterial Surface Display Systems Based on Outer Membrane Anchoring Elements from the Marine Bacterium Vibrio anguillarum ▿ ▿ †

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.02499-07

    Time course of GFP surface display in Top10/pL-U-G (A) and Top10/pW-orf1-G (B) analyzed by ELISA. Cells were cultured in LB medium and induced at 37°C, and different samples of cytoplasmic/periplasmic fraction (▪) and outer membrane fraction (□) were harvested at different time. CP, cytoplasmic/periplasmic fraction; OM, outer membrane fraction.
    Figure Legend Snippet: Time course of GFP surface display in Top10/pL-U-G (A) and Top10/pW-orf1-G (B) analyzed by ELISA. Cells were cultured in LB medium and induced at 37°C, and different samples of cytoplasmic/periplasmic fraction (▪) and outer membrane fraction (□) were harvested at different time. CP, cytoplasmic/periplasmic fraction; OM, outer membrane fraction.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Cell Culture

    Effect of induction temperature on outer membrane display and cell growth of strains Top10/pL-U-G, Top10/pW-orf1-G, and Top10/pG. OM, outer membrane fraction. Cells were cultured in LB medium and induced at 37°C (A), 30°C (B), or 25°C (C) for 12 h and followed by fractionation.
    Figure Legend Snippet: Effect of induction temperature on outer membrane display and cell growth of strains Top10/pL-U-G, Top10/pW-orf1-G, and Top10/pG. OM, outer membrane fraction. Cells were cultured in LB medium and induced at 37°C (A), 30°C (B), or 25°C (C) for 12 h and followed by fractionation.

    Techniques Used: Cell Culture, Fractionation

    Western blot analysis for the cytoplasmic/periplasmic fraction and outer membrane fraction of Top10/pG (lanes 1 and 2), Top10/pW-U-G (lanes 3 and 4), Top10/pW-orf1-G (lanes 5 and 6), Top10/pL-26La-G (lanes 7 and 8), Top10/pL-U-G (lanes 9 and 10), and Top10/pL-O-G (lanes 11 and 12) (A) and protease accessibility analysis of the fusion proteins Top10/pW-orf1-G (lane 1), Top10/pL-U-G (lane 2), and Top10/pG (lane 3) (B). CP, cytoplasmic/periplasmic fraction; OM, outer membrane fraction. Lanes M, protein molecular weight marker (MBI Fermentas).
    Figure Legend Snippet: Western blot analysis for the cytoplasmic/periplasmic fraction and outer membrane fraction of Top10/pG (lanes 1 and 2), Top10/pW-U-G (lanes 3 and 4), Top10/pW-orf1-G (lanes 5 and 6), Top10/pL-26La-G (lanes 7 and 8), Top10/pL-U-G (lanes 9 and 10), and Top10/pL-O-G (lanes 11 and 12) (A) and protease accessibility analysis of the fusion proteins Top10/pW-orf1-G (lane 1), Top10/pL-U-G (lane 2), and Top10/pG (lane 3) (B). CP, cytoplasmic/periplasmic fraction; OM, outer membrane fraction. Lanes M, protein molecular weight marker (MBI Fermentas).

    Techniques Used: Western Blot, Molecular Weight, Marker

    Time course of GFP surface display in Top10/pL-U-G (A) and Top10/pW-orf1-G (B) analyzed by Western blotting. Cells were cultured in LB medium and induced at 37°C, and different fraction samples were harvested after 21 h. OM, outer membrane fraction; CP, cytoplasmic/periplasmic fraction. Lanes M, protein molecular weight marker (MBI Fermentas).
    Figure Legend Snippet: Time course of GFP surface display in Top10/pL-U-G (A) and Top10/pW-orf1-G (B) analyzed by Western blotting. Cells were cultured in LB medium and induced at 37°C, and different fraction samples were harvested after 21 h. OM, outer membrane fraction; CP, cytoplasmic/periplasmic fraction. Lanes M, protein molecular weight marker (MBI Fermentas).

    Techniques Used: Western Blot, Cell Culture, Molecular Weight, Marker

    8) Product Images from "Balancing mcr-1 expression and bacterial survival is a delicate equilibrium between essential cellular defence mechanisms"

    Article Title: Balancing mcr-1 expression and bacterial survival is a delicate equilibrium between essential cellular defence mechanisms

    Journal: Nature Communications

    doi: 10.1038/s41467-017-02149-0

    Competition assays and G. mellonella killing models in HLCRMs. a Fitness of full-length mcr-1 , calalytically inactivated mcr-1 (E246A), and mcr-1 soluble domain. Fitness of blaTEM-1b as a negative control. b Relative fitness of wild-type mcr-1 -positive strains and their derivatives. c Relative fitness of mcr-1/ pBAD-positive E.coli TOP10 strains and their derivatives. In all fitness figures, error bars represent the SD ( n = 6). The differences in fitness were tested using non-parametric Mann–Whitney test, * indicates 0.01
    Figure Legend Snippet: Competition assays and G. mellonella killing models in HLCRMs. a Fitness of full-length mcr-1 , calalytically inactivated mcr-1 (E246A), and mcr-1 soluble domain. Fitness of blaTEM-1b as a negative control. b Relative fitness of wild-type mcr-1 -positive strains and their derivatives. c Relative fitness of mcr-1/ pBAD-positive E.coli TOP10 strains and their derivatives. In all fitness figures, error bars represent the SD ( n = 6). The differences in fitness were tested using non-parametric Mann–Whitney test, * indicates 0.01

    Techniques Used: Negative Control, MANN-WHITNEY

    TEM micrographs of untreated and treated E.coli . In a and b , TEM micrographs of untreated control cells ( E. coli TOP10 with pBAD minus mcr-1 , and E. coli TOP10 ( mcr-1 /pBAD) without l -arabinose induction, respectively); both cells are intact with a well-defined inner and outer membrane, and showed a highly homogeneous electron density in cytoplasm region ( d and e ). c TEM micrographs of mcr-1 overproducing cells; the damaging outer membrane and some completely lysed cells were observed ( f )
    Figure Legend Snippet: TEM micrographs of untreated and treated E.coli . In a and b , TEM micrographs of untreated control cells ( E. coli TOP10 with pBAD minus mcr-1 , and E. coli TOP10 ( mcr-1 /pBAD) without l -arabinose induction, respectively); both cells are intact with a well-defined inner and outer membrane, and showed a highly homogeneous electron density in cytoplasm region ( d and e ). c TEM micrographs of mcr-1 overproducing cells; the damaging outer membrane and some completely lysed cells were observed ( f )

    Techniques Used: Transmission Electron Microscopy

    9) Product Images from "Transmating: conjugative transfer of a new broad host range expression vector to various Bacillus species using a single protocol"

    Article Title: Transmating: conjugative transfer of a new broad host range expression vector to various Bacillus species using a single protocol

    Journal: BMC Microbiology

    doi: 10.1186/s12866-018-1198-4

    Steps for transfer of pBACOV-sfGFP from E. coli to Bacillus species by transmating. Triparental conjugation (transmating) requires an acceptor strain (strain of a Bacillus species of choice), a donor strain (e. g. E. coli TOP10 carrying pBACOV-sfGFP) and a helper strain ( E. coli HB101 pRK2013). After conjugation on agar plates without selection, the cells are transferred to appropriate antibiotic-containing plates (Pol concentration
    Figure Legend Snippet: Steps for transfer of pBACOV-sfGFP from E. coli to Bacillus species by transmating. Triparental conjugation (transmating) requires an acceptor strain (strain of a Bacillus species of choice), a donor strain (e. g. E. coli TOP10 carrying pBACOV-sfGFP) and a helper strain ( E. coli HB101 pRK2013). After conjugation on agar plates without selection, the cells are transferred to appropriate antibiotic-containing plates (Pol concentration

    Techniques Used: Conjugation Assay, Selection, Concentration Assay

    10) Product Images from "Improved Detection of Domoic Acid Using Covalently Immobilised Antibody Fragments"

    Article Title: Improved Detection of Domoic Acid Using Covalently Immobilised Antibody Fragments

    Journal: Marine Drugs

    doi: 10.3390/md11030881

    Comparison of expression of scFv variants in E. coli . Lanes 1,6: molecular weight markers; Lanes 2,3: soluble, insoluble 2H12 scFv produced under autoinduction conditions in E. coli BL21(DE3) for 48 h; Lanes 4,5: soluble, insoluble 2H12 scFv-cys I (autoinduction, 24 h, E. coli TOP10); Lanes 7,8: soluble, insoluble 2H12 scFv-cys II (autoinduction, 24 h, E. coli TOP10).
    Figure Legend Snippet: Comparison of expression of scFv variants in E. coli . Lanes 1,6: molecular weight markers; Lanes 2,3: soluble, insoluble 2H12 scFv produced under autoinduction conditions in E. coli BL21(DE3) for 48 h; Lanes 4,5: soluble, insoluble 2H12 scFv-cys I (autoinduction, 24 h, E. coli TOP10); Lanes 7,8: soluble, insoluble 2H12 scFv-cys II (autoinduction, 24 h, E. coli TOP10).

    Techniques Used: Expressing, Molecular Weight, Produced

    11) Product Images from "Conservation of the Low-shear Modeled Microgravity Response in Enterobacteriaceae and Analysis of the trp Genes in this Response"

    Article Title: Conservation of the Low-shear Modeled Microgravity Response in Enterobacteriaceae and Analysis of the trp Genes in this Response

    Journal: The Open Microbiology Journal

    doi: 10.2174/1874285801408010051

    Growth of Enterobacteriaceae in the RWV. Panel A : A representative growth curve for Enterobacteriaceae ( Enterobacter cloacae ATCC23355) in the RWV for the LSMMG and control conditions in LB media, 37 °C. The RWV apparatuses were set-up and operated as shown in Fig. ( 1 ) and as previously described [1, 3-5, 7]. At the indicated time points, aliquots were removed from the indicated apparatuses, serial-diluted, and plated for CFU/ml counts in triplicate. This experiment was also performed for Escherichia coli TOP10, Citrobacter freudii ATCC8090, and Serratia marcsescens ATCC14041 with equivalent results (data shown in panel B below). Panel B : Cultures of the indicated bacteria were grown in the RWV in the LSMMG and control conditions in LB media and samples processed as in panel A. For each time point, the ratio of CFU/ml LSMMG to control was calculated. The results were obtained from at least two independent cultures for each strain, and the average and standard deviation are plotted. For E. coli , the data shown here is for strain TOP10. Strains E. coli MG1655, E. coli DH5 α, and E. coli AS11 displayed results equivalent to those presented here (data not shown).
    Figure Legend Snippet: Growth of Enterobacteriaceae in the RWV. Panel A : A representative growth curve for Enterobacteriaceae ( Enterobacter cloacae ATCC23355) in the RWV for the LSMMG and control conditions in LB media, 37 °C. The RWV apparatuses were set-up and operated as shown in Fig. ( 1 ) and as previously described [1, 3-5, 7]. At the indicated time points, aliquots were removed from the indicated apparatuses, serial-diluted, and plated for CFU/ml counts in triplicate. This experiment was also performed for Escherichia coli TOP10, Citrobacter freudii ATCC8090, and Serratia marcsescens ATCC14041 with equivalent results (data shown in panel B below). Panel B : Cultures of the indicated bacteria were grown in the RWV in the LSMMG and control conditions in LB media and samples processed as in panel A. For each time point, the ratio of CFU/ml LSMMG to control was calculated. The results were obtained from at least two independent cultures for each strain, and the average and standard deviation are plotted. For E. coli , the data shown here is for strain TOP10. Strains E. coli MG1655, E. coli DH5 α, and E. coli AS11 displayed results equivalent to those presented here (data not shown).

    Techniques Used: Standard Deviation

    12) Product Images from "Phage Display of the Serpin Alpha-1 Proteinase Inhibitor Randomized at Consecutive Residues in the Reactive Centre Loop and Biopanned with or without Thrombin"

    Article Title: Phage Display of the Serpin Alpha-1 Proteinase Inhibitor Randomized at Consecutive Residues in the Reactive Centre Loop and Biopanned with or without Thrombin

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0084491

    Schematic diagrams of construction of phage display library and biopanning strategy. Panel A shows the cloning strategy employed to create T7Select10-3b phage constructs displaying API M358R or libraries displaying API with randomized RCL codons. 1) The API M358R cDNA was mobilized from pBAD-H 6 API M358R [29] by PCR that appropriately positioned EcoRI (green) and HindIII cohesive ends, and ensured that it was in-frame for subsequent insertion into the T7 Select 10-3b vector, in plasmid pUC19-API M358R. 2) Degeneracy was introduced at either P2–P1 or P7–P3 codons using a sense primer overlapping the unique PmlI site or a degenerate antisense (NNN primer) overlapping the unique SauI site. PCR products were inserted into phosphatase-treated PmlI- and SauI-restricted pUC19-API M358R and the resulting plasmid library biologically amplified in E. coli TOP10. 3) EcoRI-and HindIII-restricted total digestion products of the P2–P1 or P7–P3 plasmid libraries, respectively, were then ligated to T7Select10-3b vector arms. 4) The recombinant T7Select10-3b library, containing either the API P2–P1 randomized or the API M358R P7–P3 randomized inserts, was then packaged into phages to create the respective phage display libraries. Pink, light blue, yellow, and purple API-encoding inserts (between steps 2 and 3) indicate the encoding of different variants, and the correspondingly coloured phages (step 4) represent their displayed products. Panel B shows the biopanning procedure. 1) A phage lysate produced by infection of E. coli BLT5615 with a T7Select10-3b API library was reacted with thrombin in solution (green squares), which bound to some displayed sequences (e.g. blue but not yellow or pink). 2) A biotinylated antibody specific to thrombin (Y-shaped symbols) was added to the thrombin-phages mixture, reacting with thrombin bound to phages via API-thrombin serpin-enzyme complexes. 3) Streptavidin-coated magnetic beads (yellow S shown on red circular symbols) were added to the mixture, reacting preferentially with antibody-thrombin-phage complexes. 4) Magnetic bead-streptavidin-thrombin-phage complexes were concentrated using a magnet. 5) After washing, bead assemblies were used to directly infect a fresh culture of E. coli BLT5615 to start the next round of biopanning.
    Figure Legend Snippet: Schematic diagrams of construction of phage display library and biopanning strategy. Panel A shows the cloning strategy employed to create T7Select10-3b phage constructs displaying API M358R or libraries displaying API with randomized RCL codons. 1) The API M358R cDNA was mobilized from pBAD-H 6 API M358R [29] by PCR that appropriately positioned EcoRI (green) and HindIII cohesive ends, and ensured that it was in-frame for subsequent insertion into the T7 Select 10-3b vector, in plasmid pUC19-API M358R. 2) Degeneracy was introduced at either P2–P1 or P7–P3 codons using a sense primer overlapping the unique PmlI site or a degenerate antisense (NNN primer) overlapping the unique SauI site. PCR products were inserted into phosphatase-treated PmlI- and SauI-restricted pUC19-API M358R and the resulting plasmid library biologically amplified in E. coli TOP10. 3) EcoRI-and HindIII-restricted total digestion products of the P2–P1 or P7–P3 plasmid libraries, respectively, were then ligated to T7Select10-3b vector arms. 4) The recombinant T7Select10-3b library, containing either the API P2–P1 randomized or the API M358R P7–P3 randomized inserts, was then packaged into phages to create the respective phage display libraries. Pink, light blue, yellow, and purple API-encoding inserts (between steps 2 and 3) indicate the encoding of different variants, and the correspondingly coloured phages (step 4) represent their displayed products. Panel B shows the biopanning procedure. 1) A phage lysate produced by infection of E. coli BLT5615 with a T7Select10-3b API library was reacted with thrombin in solution (green squares), which bound to some displayed sequences (e.g. blue but not yellow or pink). 2) A biotinylated antibody specific to thrombin (Y-shaped symbols) was added to the thrombin-phages mixture, reacting with thrombin bound to phages via API-thrombin serpin-enzyme complexes. 3) Streptavidin-coated magnetic beads (yellow S shown on red circular symbols) were added to the mixture, reacting preferentially with antibody-thrombin-phage complexes. 4) Magnetic bead-streptavidin-thrombin-phage complexes were concentrated using a magnet. 5) After washing, bead assemblies were used to directly infect a fresh culture of E. coli BLT5615 to start the next round of biopanning.

    Techniques Used: Clone Assay, Construct, Polymerase Chain Reaction, Plasmid Preparation, Amplification, Recombinant, Produced, Infection, Magnetic Beads

    13) Product Images from "Quantification of the gene silencing performances of rationally-designed synthetic small RNAs"

    Article Title: Quantification of the gene silencing performances of rationally-designed synthetic small RNAs

    Journal: Systems and Synthetic Biology

    doi: 10.1007/s11693-015-9177-7

    Silencing results for RFP and GFP expressed by the Plux-RG and Plux-GR synthetic operons in the TOP10 strain. a Silencing of the target gene (RFP) and the non-target gene (GFP) via the silencing device sRFP. b Unspecific silencing of RFP and GFP, in the
    Figure Legend Snippet: Silencing results for RFP and GFP expressed by the Plux-RG and Plux-GR synthetic operons in the TOP10 strain. a Silencing of the target gene (RFP) and the non-target gene (GFP) via the silencing device sRFP. b Unspecific silencing of RFP and GFP, in the

    Techniques Used:

    Lactate dehydrogenase assay results for sLDH characterization in TOP10 and W. Specific enzymatic activity of LdhA in the strain without sRNA, in the strain with an unspecific sRNA (sRFP) and in the strain with the sRNA targeting LdhA (sLDH). Bars represent
    Figure Legend Snippet: Lactate dehydrogenase assay results for sLDH characterization in TOP10 and W. Specific enzymatic activity of LdhA in the strain without sRNA, in the strain with an unspecific sRNA (sRFP) and in the strain with the sRNA targeting LdhA (sLDH). Bars represent

    Techniques Used: Lactate Dehydrogenase Assay, Activity Assay

    Silencing results for RFP or GFP expressed by a single-gene cassette driven by P lux (Plux-R or Plux-G). a Specific silencing of the target gene (RFP) via sRFP in TOP10 and W. b Unspecific silencing of RFP or GFP via different sRNAs in TOP10 and W. Bars
    Figure Legend Snippet: Silencing results for RFP or GFP expressed by a single-gene cassette driven by P lux (Plux-R or Plux-G). a Specific silencing of the target gene (RFP) via sRFP in TOP10 and W. b Unspecific silencing of RFP or GFP via different sRNAs in TOP10 and W. Bars

    Techniques Used:

    Silencing results for RFP expressed by a single-gene cassette driven by BBa_J23101 (J101-R). a Specific silencing of the target gene (RFP) via the specific silencing device (sRFP) in TOP10 and W. b Experimental S cell results and values predicted by the
    Figure Legend Snippet: Silencing results for RFP expressed by a single-gene cassette driven by BBa_J23101 (J101-R). a Specific silencing of the target gene (RFP) via the specific silencing device (sRFP) in TOP10 and W. b Experimental S cell results and values predicted by the

    Techniques Used:

    RFP silencing as a function of target mRNA and sRNA levels in TOP10 with the Plux-R construct. Data points represent average S cell values in presence of sRFP in three different copy number conditions (corresponding to three different sRFP levels), as
    Figure Legend Snippet: RFP silencing as a function of target mRNA and sRNA levels in TOP10 with the Plux-R construct. Data points represent average S cell values in presence of sRFP in three different copy number conditions (corresponding to three different sRFP levels), as

    Techniques Used: Construct

    14) Product Images from "A synthetic oligo library and sequencing approach reveals an insulation mechanism encoded within bacterial σ54 promoters"

    Article Title: A synthetic oligo library and sequencing approach reveals an insulation mechanism encoded within bacterial σ54 promoters

    Journal: bioRxiv

    doi: 10.1101/086108

    The glnKp σ 54 promoter can down-regulate another promoter positioned upstream. (A) Synthetic enhancer design showing the different UAS and σ 54 promoter combinations used in the experiment. (B) Left: mCherry expression with enhancer switched to “on’’ (NtrC induced), showing varying response for each promoter. Note that for the dual UAS-σ 70 promoter glnAp1 there is expression with the “no promoter’’ control. Right: mCherry expression for enhancers switched to “off’’ (NtrC not induced), showing “on’’ behavior for all enhancers containing the dual UAS-σ 70 promoter, except for the enhancer with the glnKp. Error level for the mean expression values are provided in Table S3 . (C) Flow cytometry data comparing mCherry fluorescence for the glnKp strain in the E. coli TOP10 strain (purple) and in the σ 54 knock-out strain (TOP10:Δ rpoN , orange). (D) qPCR data showing a reduction in mRNA level in the silenced strain (right) as compared with non-silenced strains (middle) and the no-σ 70 control (left). (E) plate-reader data showing rescue of mCherry fluorescence when the orientation of the glnKp is flipped relative to the upstream σ 70 promoter. Associated with Figure S1 , Table S1 , S2 , and S3 .
    Figure Legend Snippet: The glnKp σ 54 promoter can down-regulate another promoter positioned upstream. (A) Synthetic enhancer design showing the different UAS and σ 54 promoter combinations used in the experiment. (B) Left: mCherry expression with enhancer switched to “on’’ (NtrC induced), showing varying response for each promoter. Note that for the dual UAS-σ 70 promoter glnAp1 there is expression with the “no promoter’’ control. Right: mCherry expression for enhancers switched to “off’’ (NtrC not induced), showing “on’’ behavior for all enhancers containing the dual UAS-σ 70 promoter, except for the enhancer with the glnKp. Error level for the mean expression values are provided in Table S3 . (C) Flow cytometry data comparing mCherry fluorescence for the glnKp strain in the E. coli TOP10 strain (purple) and in the σ 54 knock-out strain (TOP10:Δ rpoN , orange). (D) qPCR data showing a reduction in mRNA level in the silenced strain (right) as compared with non-silenced strains (middle) and the no-σ 70 control (left). (E) plate-reader data showing rescue of mCherry fluorescence when the orientation of the glnKp is flipped relative to the upstream σ 70 promoter. Associated with Figure S1 , Table S1 , S2 , and S3 .

    Techniques Used: Expressing, Flow Cytometry, Fluorescence, Knock-Out, Real-time Polymerase Chain Reaction

    15) Product Images from "Regulated Expression of the Escherichia coli lepB Gene as a Tool for Cellular Testing of Antimicrobial Compounds That Inhibit Signal Peptidase I In Vitro"

    Article Title: Regulated Expression of the Escherichia coli lepB Gene as a Tool for Cellular Testing of Antimicrobial Compounds That Inhibit Signal Peptidase I In Vitro

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.46.11.3549-3554.2002

    Growth of E. coli TOP10 bearing pMB2 was investigated in LB medium (LB) and LB medium supplemented with 20 μg of Pbn per ml (LB-Pbn), 100 μM penem (LB-P), or both Pbn and penem (LB-Pbn-P). Growth was measured as the increase in optical density at 600 nm after 20 h of incubation. The initial optical density of the inoculum was 0.1. Experiments were performed in duplicate.
    Figure Legend Snippet: Growth of E. coli TOP10 bearing pMB2 was investigated in LB medium (LB) and LB medium supplemented with 20 μg of Pbn per ml (LB-Pbn), 100 μM penem (LB-P), or both Pbn and penem (LB-Pbn-P). Growth was measured as the increase in optical density at 600 nm after 20 h of incubation. The initial optical density of the inoculum was 0.1. Experiments were performed in duplicate.

    Techniques Used: Incubation

    16) Product Images from "Outer Membrane Proteins A (OmpA) and X (OmpX) Are Essential for Basolateral Invasion of Cronobacter sakazakii ▿"

    Article Title: Outer Membrane Proteins A (OmpA) and X (OmpX) Are Essential for Basolateral Invasion of Cronobacter sakazakii ▿

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.02498-09

    Inhibition of C. sakazakii ATCC 29544 (WT) invasion of Caco-2 cells by pretreatment with purified proteins. (A) C. sakazakii recombinant OmpA and OmpX were overexpressed in E. coli TOP10 and isolated using metal affinity resin. The purified proteins were
    Figure Legend Snippet: Inhibition of C. sakazakii ATCC 29544 (WT) invasion of Caco-2 cells by pretreatment with purified proteins. (A) C. sakazakii recombinant OmpA and OmpX were overexpressed in E. coli TOP10 and isolated using metal affinity resin. The purified proteins were

    Techniques Used: Inhibition, Purification, Recombinant, Isolation

    17) Product Images from "An Escherichia coli Expression Assay and Screen for Human Immunodeficiency Virus Protease Variants with Decreased Susceptibility to Indinavir"

    Article Title: An Escherichia coli Expression Assay and Screen for Human Immunodeficiency Virus Protease Variants with Decreased Susceptibility to Indinavir

    Journal: Antimicrobial Agents and Chemotherapy

    doi:

    Construction and screening of a library of HIV protease (PR) variants. Error-prone PCR was used to introduce dispersed mutations within the HIV protease gene. This mutagenized population of DNAs was ligated into the E. coli expression vector pTrcHisC (Invitrogen) along with unmutagenized DNA encoding the entire HIV reverse transcriptase (RT) gene. This plasmid library was used to transform E. coli Top10 cells (Invitrogen), and single colonies were distributed into individual microplate wells containing growth media and the protease inhibitor indinavir. IPTG was added to induce expression, cells were made permeable by freezing and thawing, and reverse transcriptase activity was assayed as described in Materials and Methods. DrugS, drug susceptible; DrugR, drug resistant.
    Figure Legend Snippet: Construction and screening of a library of HIV protease (PR) variants. Error-prone PCR was used to introduce dispersed mutations within the HIV protease gene. This mutagenized population of DNAs was ligated into the E. coli expression vector pTrcHisC (Invitrogen) along with unmutagenized DNA encoding the entire HIV reverse transcriptase (RT) gene. This plasmid library was used to transform E. coli Top10 cells (Invitrogen), and single colonies were distributed into individual microplate wells containing growth media and the protease inhibitor indinavir. IPTG was added to induce expression, cells were made permeable by freezing and thawing, and reverse transcriptase activity was assayed as described in Materials and Methods. DrugS, drug susceptible; DrugR, drug resistant.

    Techniques Used: Polymerase Chain Reaction, Introduce, Expressing, Plasmid Preparation, Protease Inhibitor, Activity Assay

    18) Product Images from "Balancing mcr-1 expression and bacterial survival is a delicate equilibrium between essential cellular defence mechanisms"

    Article Title: Balancing mcr-1 expression and bacterial survival is a delicate equilibrium between essential cellular defence mechanisms

    Journal: Nature Communications

    doi: 10.1038/s41467-017-02149-0

    Competition assays and G. mellonella killing models in HLCRMs. a Fitness of full-length mcr-1 , calalytically inactivated mcr-1 (E246A), and mcr-1 soluble domain. Fitness of blaTEM-1b as a negative control. b Relative fitness of wild-type mcr-1 -positive strains and their derivatives. c Relative fitness of mcr-1/ pBAD-positive E.coli TOP10 strains and their derivatives. In all fitness figures, error bars represent the SD ( n = 6). The differences in fitness were tested using non-parametric Mann–Whitney test, * indicates 0.01
    Figure Legend Snippet: Competition assays and G. mellonella killing models in HLCRMs. a Fitness of full-length mcr-1 , calalytically inactivated mcr-1 (E246A), and mcr-1 soluble domain. Fitness of blaTEM-1b as a negative control. b Relative fitness of wild-type mcr-1 -positive strains and their derivatives. c Relative fitness of mcr-1/ pBAD-positive E.coli TOP10 strains and their derivatives. In all fitness figures, error bars represent the SD ( n = 6). The differences in fitness were tested using non-parametric Mann–Whitney test, * indicates 0.01

    Techniques Used: Negative Control, MANN-WHITNEY

    TEM micrographs of untreated and treated E.coli . In a and b , TEM micrographs of untreated control cells ( E. coli TOP10 with pBAD minus mcr-1 , and E. coli TOP10 ( mcr-1 /pBAD) without l -arabinose induction, respectively); both cells are intact with a well-defined inner and outer membrane, and showed a highly homogeneous electron density in cytoplasm region ( d and e ). c TEM micrographs of mcr-1 overproducing cells; the damaging outer membrane and some completely lysed cells were observed ( f )
    Figure Legend Snippet: TEM micrographs of untreated and treated E.coli . In a and b , TEM micrographs of untreated control cells ( E. coli TOP10 with pBAD minus mcr-1 , and E. coli TOP10 ( mcr-1 /pBAD) without l -arabinose induction, respectively); both cells are intact with a well-defined inner and outer membrane, and showed a highly homogeneous electron density in cytoplasm region ( d and e ). c TEM micrographs of mcr-1 overproducing cells; the damaging outer membrane and some completely lysed cells were observed ( f )

    Techniques Used: Transmission Electron Microscopy

    19) Product Images from "Genetic Evidence for Functional Interaction of the Escherichia coli Signal Recognition Particle Receptor with Acidic Lipids in Vivo *"

    Article Title: Genetic Evidence for Functional Interaction of the Escherichia coli Signal Recognition Particle Receptor with Acidic Lipids in Vivo *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.140921

    Effect of PgsA overexpression on lipid composition in vivo . A , thin layer chromatography separation of phospholipid standards. B, E. coli Top10 harboring plasmid-encoded variants of PgsA or empty vectors were labeled with [ 32 P]phosphoric acid at a final concentration of 1 μCi/ml and grown for 3 h at 37 °C. Lipids were extracted and separated by thin layer chromatography. C , quantitation of the phospholipid composition (moi % of lipid phosphorus calculated from radioactivity of each spot, see “Experimental Procedures”). The data shown are mean values, and the standard deviations are calculated from two or three different batches of cultures.
    Figure Legend Snippet: Effect of PgsA overexpression on lipid composition in vivo . A , thin layer chromatography separation of phospholipid standards. B, E. coli Top10 harboring plasmid-encoded variants of PgsA or empty vectors were labeled with [ 32 P]phosphoric acid at a final concentration of 1 μCi/ml and grown for 3 h at 37 °C. Lipids were extracted and separated by thin layer chromatography. C , quantitation of the phospholipid composition (moi % of lipid phosphorus calculated from radioactivity of each spot, see “Experimental Procedures”). The data shown are mean values, and the standard deviations are calculated from two or three different batches of cultures.

    Techniques Used: Over Expression, In Vivo, Thin Layer Chromatography, Plasmid Preparation, Labeling, Concentration Assay, Quantitation Assay, Radioactivity

    20) Product Images from "New Plasmid-Mediated Quinolone Resistance Gene, qnrC, Found in a Clinical Isolate of Proteus mirabilis ▿"

    Article Title: New Plasmid-Mediated Quinolone Resistance Gene, qnrC, Found in a Clinical Isolate of Proteus mirabilis ▿

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.01400-08

    Genetic environment of the qnrC gene in the 4.4-kb HindIII fragment of plasmid pHS11. E. coli TOP10 cells containing pHS11 had a ciprofloxacin MIC of 0.125 μg/ml and a levofloxacin MIC of 0.19 μg/ml. IS Pmi1 is enclosed by two inverted
    Figure Legend Snippet: Genetic environment of the qnrC gene in the 4.4-kb HindIII fragment of plasmid pHS11. E. coli TOP10 cells containing pHS11 had a ciprofloxacin MIC of 0.125 μg/ml and a levofloxacin MIC of 0.19 μg/ml. IS Pmi1 is enclosed by two inverted

    Techniques Used: Plasmid Preparation

    21) Product Images from "Novel Dehalogenase Mechanism for 2,3-Dichloro-1-Propanol Utilization in Pseudomonas putida Strain MC4"

    Article Title: Novel Dehalogenase Mechanism for 2,3-Dichloro-1-Propanol Utilization in Pseudomonas putida Strain MC4

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.00760-12

    Coomassie stain (A) and heme stain (B) of an SDS-PAGE gel containing recombinant DppA expressed in E. coli strains JCB712 (lanes 1 and 2) and TOP10 (lane 3). Different concentrations of arabinose were used for induction: lane 1, 0.002%; lanes 2 and 3,
    Figure Legend Snippet: Coomassie stain (A) and heme stain (B) of an SDS-PAGE gel containing recombinant DppA expressed in E. coli strains JCB712 (lanes 1 and 2) and TOP10 (lane 3). Different concentrations of arabinose were used for induction: lane 1, 0.002%; lanes 2 and 3,

    Techniques Used: Staining, SDS Page, Recombinant

    22) Product Images from "An engineered small RNA-mediated genetic switch based on a ribozyme expression platform"

    Article Title: An engineered small RNA-mediated genetic switch based on a ribozyme expression platform

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkt253

    Structure–function relationship of ta RNAs with modified seed regions. ( A ) Structural modifications were introduced into the single-stranded seed region by either reducing (V1-M5) or extending (V1-M4) the amount of complementary bases to the hybridization domain (blue) at the 3′ terminus of the ta RNA. The variants V1-M4 and V1-M5 are both fully complementary to the ta RNA responsive element. The site of 5′-processing is marked by an arrow head. All ta RNA constructs were engineered under control of an IPTG-inducible promoter. ( B ) The influence of the ta RNAs shown in A on TR-HHR activity was examined. Bacterial cultures of the E. coli Top10 strain were induced with 20 µM arabinose and 1 mM IPTG. Transformants were cultivated in LB medium at 37°C, and reporter gene expression of outgrown bacterial cultures was measured. Error bars represent the standard deviation of experiments performed in triplicates.
    Figure Legend Snippet: Structure–function relationship of ta RNAs with modified seed regions. ( A ) Structural modifications were introduced into the single-stranded seed region by either reducing (V1-M5) or extending (V1-M4) the amount of complementary bases to the hybridization domain (blue) at the 3′ terminus of the ta RNA. The variants V1-M4 and V1-M5 are both fully complementary to the ta RNA responsive element. The site of 5′-processing is marked by an arrow head. All ta RNA constructs were engineered under control of an IPTG-inducible promoter. ( B ) The influence of the ta RNAs shown in A on TR-HHR activity was examined. Bacterial cultures of the E. coli Top10 strain were induced with 20 µM arabinose and 1 mM IPTG. Transformants were cultivated in LB medium at 37°C, and reporter gene expression of outgrown bacterial cultures was measured. Error bars represent the standard deviation of experiments performed in triplicates.

    Techniques Used: Modification, Hybridization, Construct, Activity Assay, Expressing, Standard Deviation

    Influence of truncated ta RNAs. ( A ) The helical region outside of the hybridization domain was shortened. The variants V1-M6 and V1-M7 are both fully complementary to the ta RNA responsive element. The site of 5′-processing is marked by an arrow head. ( B ) The influence of the ta RNAs shown in A on the TR-HHR activity was examined. Bacterial cultures of the E. coli Top10 strain were induced with 20 µM arabinose and 1 mM IPTG. Transformants were cultivated in LB medium at 37°C, and eGFP expression of outgrown bacterial cultures was measured. Error bars represent the standard deviation of experiments performed in triplicates.
    Figure Legend Snippet: Influence of truncated ta RNAs. ( A ) The helical region outside of the hybridization domain was shortened. The variants V1-M6 and V1-M7 are both fully complementary to the ta RNA responsive element. The site of 5′-processing is marked by an arrow head. ( B ) The influence of the ta RNAs shown in A on the TR-HHR activity was examined. Bacterial cultures of the E. coli Top10 strain were induced with 20 µM arabinose and 1 mM IPTG. Transformants were cultivated in LB medium at 37°C, and eGFP expression of outgrown bacterial cultures was measured. Error bars represent the standard deviation of experiments performed in triplicates.

    Techniques Used: Hybridization, Activity Assay, Expressing, Standard Deviation

    The sRNA-mediated repression of translation depends on the formation of the RNA–RNA hybrid structure. ( A ) Schematic illustration of artificial ta RNA variants with reduced number of complementary nucleobases to the ta RNA responsive element was constructed. The constructs V1-M1 and V1-M2 display partial hybridization capability (blue nucleobases), whereas the construct V1-M3 is non-complementary at all. The site of 5′-processing is marked by an arrow head. All ta RNA constructs were engineered under control of an IPTG-inducible promoter. ( B ) The influence of the ta RNAs shown in (A) on the TR-HHR activity was examined. Bacterial cultures of the E. coli Top10 strain were induced with 20 µM arabinose and 1 mM IPTG. Transformants were cultivated in LB medium at 37°C, and eGFP expression of outgrown bacterial cultures was measured. Error bars represent the standard deviation of experiments performed in triplicates. ( C ) The inactivation of the TR-HHR results in non-detectable eGFP expression levels and was not influenced by the co-expressed ta RNAs Crl, V1 and V1-M3. The assay was performed as described earlier in the text. ( D ) Analysis of eGFP mRNA and ta RNA levels by semi-quantitative RT-PCR. Bacterial cultures were induced with 20 µM arabinose and 1 mM IPTG. Results indicate a high excess of ta RNA over eGFP mRNA transcripts and an accumulation of ta RNA V1 through the 5′-processing reaction of the HHR. ( E–G ) Heat maps of observed repression of translation by the constructs (E) V1, (F) V1-M1 and (G) V1-M2 as function of transcriptional induction of the reporter construct and the ta RNAs. Data were normalized to the construct V1-M3. Transcription of the reporter gene was induced by the addition of 20, 80 and 320 µM arabinose, and transcription of the ta RNA variants was induced by the addition of 0, 100, 333 and 1000 µM IPTG. The translational repressed state is indicated in blue, whereas the unrepressed state is shown red. Bacterial cultures of the E. coli Top10 strain were induced with arabinose and IPTG as indicated. Transformants were cultivated in LB medium at 37°C, and eGFP expression of outgrown bacterial cultures was determined.
    Figure Legend Snippet: The sRNA-mediated repression of translation depends on the formation of the RNA–RNA hybrid structure. ( A ) Schematic illustration of artificial ta RNA variants with reduced number of complementary nucleobases to the ta RNA responsive element was constructed. The constructs V1-M1 and V1-M2 display partial hybridization capability (blue nucleobases), whereas the construct V1-M3 is non-complementary at all. The site of 5′-processing is marked by an arrow head. All ta RNA constructs were engineered under control of an IPTG-inducible promoter. ( B ) The influence of the ta RNAs shown in (A) on the TR-HHR activity was examined. Bacterial cultures of the E. coli Top10 strain were induced with 20 µM arabinose and 1 mM IPTG. Transformants were cultivated in LB medium at 37°C, and eGFP expression of outgrown bacterial cultures was measured. Error bars represent the standard deviation of experiments performed in triplicates. ( C ) The inactivation of the TR-HHR results in non-detectable eGFP expression levels and was not influenced by the co-expressed ta RNAs Crl, V1 and V1-M3. The assay was performed as described earlier in the text. ( D ) Analysis of eGFP mRNA and ta RNA levels by semi-quantitative RT-PCR. Bacterial cultures were induced with 20 µM arabinose and 1 mM IPTG. Results indicate a high excess of ta RNA over eGFP mRNA transcripts and an accumulation of ta RNA V1 through the 5′-processing reaction of the HHR. ( E–G ) Heat maps of observed repression of translation by the constructs (E) V1, (F) V1-M1 and (G) V1-M2 as function of transcriptional induction of the reporter construct and the ta RNAs. Data were normalized to the construct V1-M3. Transcription of the reporter gene was induced by the addition of 20, 80 and 320 µM arabinose, and transcription of the ta RNA variants was induced by the addition of 0, 100, 333 and 1000 µM IPTG. The translational repressed state is indicated in blue, whereas the unrepressed state is shown red. Bacterial cultures of the E. coli Top10 strain were induced with arabinose and IPTG as indicated. Transformants were cultivated in LB medium at 37°C, and eGFP expression of outgrown bacterial cultures was determined.

    Techniques Used: Construct, Hybridization, Activity Assay, Expressing, Standard Deviation, Quantitative RT-PCR

    Engineering of TR-HHR-eGFP fusions and artificial ta RNAs. ( A ) The reporter gene, in which cleavage of the TR-HHR controls translation initiation of eGFP, was transcriptionally controlled by an arabinose-inducible P BAD promoter. The secondary structure of the highly modular HHR-based genetic switches is shown. The TR-HHR serves as the expression platform to which multiple functional RNA domains are attached. Control of translation initiation is obtained through sequestration of the RBS (gray nucleotides) by an extended stem 1. A domain harboring a ta RNA responsive element (blue) can be attached to stem 3. The YUNR motif located in the loop of stem 3 is shaded in gray. ( B ) The generation of the artificial ta RNA V1 is transcriptionally controlled by an IPTG-inducible promoter. Previous studies reported that extended 5′ single-stranded regions impede ta RNA function. Therefore, a 5P-HHR was attached to the ta RNA for 5′-processing. The autocatalytic cleavage reaction (marked by an arrow head) results in a single-stranded region made-up of 17 non-complementary nucleotides. Within the construct V1i, the 5′-processing reaction is eliminated by the A to G point-mutation within the catalytic core of the 5P-HHR. ( C ) The hybrid complex between the ta RNA responsive element of the TR-HHR and the processed ta RNA V1 is shown. ( D ) The influence of the ta RNAs V1 and V1i in comparison with a control RNA (Crl) with no complementarity to the TR-HHR was examined. Bacterial cultures of the E. coli Top10 strain were induced with 20 µM arabinose and 1 mM IPTG. Transformants were cultivated in LB medium at 37°C, and reporter gene expression of outgrown bacterial cultures was measured. Errors bars represent the standard deviation of experiments performed in triplicates.
    Figure Legend Snippet: Engineering of TR-HHR-eGFP fusions and artificial ta RNAs. ( A ) The reporter gene, in which cleavage of the TR-HHR controls translation initiation of eGFP, was transcriptionally controlled by an arabinose-inducible P BAD promoter. The secondary structure of the highly modular HHR-based genetic switches is shown. The TR-HHR serves as the expression platform to which multiple functional RNA domains are attached. Control of translation initiation is obtained through sequestration of the RBS (gray nucleotides) by an extended stem 1. A domain harboring a ta RNA responsive element (blue) can be attached to stem 3. The YUNR motif located in the loop of stem 3 is shaded in gray. ( B ) The generation of the artificial ta RNA V1 is transcriptionally controlled by an IPTG-inducible promoter. Previous studies reported that extended 5′ single-stranded regions impede ta RNA function. Therefore, a 5P-HHR was attached to the ta RNA for 5′-processing. The autocatalytic cleavage reaction (marked by an arrow head) results in a single-stranded region made-up of 17 non-complementary nucleotides. Within the construct V1i, the 5′-processing reaction is eliminated by the A to G point-mutation within the catalytic core of the 5P-HHR. ( C ) The hybrid complex between the ta RNA responsive element of the TR-HHR and the processed ta RNA V1 is shown. ( D ) The influence of the ta RNAs V1 and V1i in comparison with a control RNA (Crl) with no complementarity to the TR-HHR was examined. Bacterial cultures of the E. coli Top10 strain were induced with 20 µM arabinose and 1 mM IPTG. Transformants were cultivated in LB medium at 37°C, and reporter gene expression of outgrown bacterial cultures was measured. Errors bars represent the standard deviation of experiments performed in triplicates.

    Techniques Used: Expressing, Functional Assay, Construct, Mutagenesis, Standard Deviation

    23) Product Images from "The Pseudomonas aeruginosa Secreted Protein PA2934 Decreases Apical Membrane Expression of the Cystic Fibrosis Transmembrane Conductance Regulator ▿"

    Article Title: The Pseudomonas aeruginosa Secreted Protein PA2934 Decreases Apical Membrane Expression of the Cystic Fibrosis Transmembrane Conductance Regulator ▿

    Journal: Infection and Immunity

    doi: 10.1128/IAI.00338-07

    PA2934 is sufficient for Cif activity. (A) Coomassie blue-stained SDS-PAGE gel of supernatant from E. coli Top10 cells carrying pDPM73 (PA2934-His) and of purified PA2934-His. Ten micrograms of protein from each sample was resolved via SDS-PAGE and stained
    Figure Legend Snippet: PA2934 is sufficient for Cif activity. (A) Coomassie blue-stained SDS-PAGE gel of supernatant from E. coli Top10 cells carrying pDPM73 (PA2934-His) and of purified PA2934-His. Ten micrograms of protein from each sample was resolved via SDS-PAGE and stained

    Techniques Used: Activity Assay, Staining, SDS Page, Purification

    24) Product Images from "Cloning and Transfer of the Salmonella Pathogenicity Island 2 Type III Secretion System for Studies of a Range of Gram-Negative Genera ▿ Pathogenicity Island 2 Type III Secretion System for Studies of a Range of Gram-Negative Genera ▿ †"

    Article Title: Cloning and Transfer of the Salmonella Pathogenicity Island 2 Type III Secretion System for Studies of a Range of Gram-Negative Genera ▿ Pathogenicity Island 2 Type III Secretion System for Studies of a Range of Gram-Negative Genera ▿ †

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.00952-07

    Western blot analysis of SseB expression in different bacterial strains containing R995 + SPI-2 grown in MgM 10 and MgM 8. (A) Total cell lysates were obtained from S. enterica serovar Typhimurium and E. coli strains containing either R995 (R) or R995 + SPI-2 (R + SPI-2) grown in the indicated medium, and equivalent amounts of these lysates were analyzed via SDS-PAGE, Western blotting, and probing with anti-SseB antisera. Lanes numbered 1 to 4 indicate four separate isolates of the strain TOP10 (R + SPI-2). MgM 10 and MgM 8 indicate media used for SPI-2 noninducing and inducing conditions, respectively. WT, wild type. (B) An analysis identical to that described for panel A was performed for other gram-negative strains containing R995 (R) or R995 + SPI-2 (R + SPI-2). The lane marked “+” contains a sample from the same batch of lysate obtained from the strain S. enterica serovar Typhimurium ΔSPI-2 (R + SPI-2) as a positive control. P.a. , P. aeruginosa .
    Figure Legend Snippet: Western blot analysis of SseB expression in different bacterial strains containing R995 + SPI-2 grown in MgM 10 and MgM 8. (A) Total cell lysates were obtained from S. enterica serovar Typhimurium and E. coli strains containing either R995 (R) or R995 + SPI-2 (R + SPI-2) grown in the indicated medium, and equivalent amounts of these lysates were analyzed via SDS-PAGE, Western blotting, and probing with anti-SseB antisera. Lanes numbered 1 to 4 indicate four separate isolates of the strain TOP10 (R + SPI-2). MgM 10 and MgM 8 indicate media used for SPI-2 noninducing and inducing conditions, respectively. WT, wild type. (B) An analysis identical to that described for panel A was performed for other gram-negative strains containing R995 (R) or R995 + SPI-2 (R + SPI-2). The lane marked “+” contains a sample from the same batch of lysate obtained from the strain S. enterica serovar Typhimurium ΔSPI-2 (R + SPI-2) as a positive control. P.a. , P. aeruginosa .

    Techniques Used: Western Blot, Expressing, SDS Page, Positive Control

    25) Product Images from "Directed evolution of an orthogonal nucleoside analog kinase via fluorescence-activated cell sorting"

    Article Title: Directed evolution of an orthogonal nucleoside analog kinase via fluorescence-activated cell sorting

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp400

    Schematic overview of the kinase library screening assay with fluorescent NAs. Upon incubation of bacteria with the target fNA, cells that carry a functional kinase variant for the specific substrate will accumulate the fNA monophosphate. Hosts cells with the highest fluorescence intensity can be identified and isolated by fluorescence-activated cell sorting (FACS) for subsequent characterization of the library member or, if desirable, another round of directed evolution. For concept validation, E. coli TOP10 expressing exogenous Dm dNK with thymidine kinase activity (+kinase) was compared to host expressing thymidine kinase-deficient human dCK (−kinase). The histogram shows the 20- to 30-fold increase in cellular fluorescence upon incubation with fT in the presence of the exogenous thymidine kinase activity.
    Figure Legend Snippet: Schematic overview of the kinase library screening assay with fluorescent NAs. Upon incubation of bacteria with the target fNA, cells that carry a functional kinase variant for the specific substrate will accumulate the fNA monophosphate. Hosts cells with the highest fluorescence intensity can be identified and isolated by fluorescence-activated cell sorting (FACS) for subsequent characterization of the library member or, if desirable, another round of directed evolution. For concept validation, E. coli TOP10 expressing exogenous Dm dNK with thymidine kinase activity (+kinase) was compared to host expressing thymidine kinase-deficient human dCK (−kinase). The histogram shows the 20- to 30-fold increase in cellular fluorescence upon incubation with fT in the presence of the exogenous thymidine kinase activity.

    Techniques Used: Library Screening, Incubation, Functional Assay, Variant Assay, Fluorescence, Isolation, FACS, Expressing, Activity Assay

    Related Articles

    Clone Assay:

    Article Title: Buwchitin: A Ruminal Peptide with Antimicrobial Potential against Enterococcus faecalis
    Article Snippet: .. E. coli TOP10 (Invitrogen, Carlsbad CA, USA) was used for cloning (to host expression vectors for protein expression). .. The pTrcHis TOPO® vector (Invitrogen, Carlsbad, CA, USA) was used to clone polymerase chain reaction (PCR) products for protein expression.

    Article Title: A Plasmid-Borne System To Assess the Excision and Integration of Staphylococcal Cassette Chromosome mec Mediated by CcrA and CcrB
    Article Snippet: .. E. coli TOP10 (Invitrogen, Carlsbad, CA) was used for gene cloning. .. The antibiotics and concentrations used were as follows: 100 μg/ml ampicillin (Ap) for selection of E. coli strains following transformation and 10 μg/ml chloramphenicol (Cml) and 5 μg/ml tetracycline (Tet) for selection of S. aureus strains following electroporation ( ).

    Acetylene Reduction Assay:

    Article Title: Novel Bacterial Surface Display Systems Based on Outer Membrane Anchoring Elements from the Marine Bacterium Vibrio anguillarum ▿ ▿ †
    Article Snippet: .. E. coli Top10 [F− mcrA Δ( mrr-hsdRMS-mcrBC ) φ80 lacZ ΔM15 Δ lacX74 deoR recA1 araD139 Δ( ara-leu ) 7697 galU galK rpsL (Strr ) endA1 nupG ] (Invitrogen, Carlsbad, CA) was used as the host for recombinant plasmid construction and GFP surface display. ..

    Purification:

    Article Title: Transfer of KPC-2 Carbapenemase from Klebsiella pneumoniae to Escherichia coli in a Patient: First Case in Europe ▿
    Article Snippet: .. Purified plasmids from K. pneumoniae and E. coli were transformed into an E. coli Top10 (Invitrogen) recipient strain and selected on plates containing 100 mg/liter ampicillin; screening for bla KPC -positive colonies showed that, in both cases, E. coli Top10 was transformed with a 20-kb plasmid, which encoded KPC-2, bla TEM-1 , and bla OXA-9 , and not bla VIM-1 , which was located on a different plasmid. .. Plasmids from the transformed colonies were purified and digested with the HindIII and EcoRI restriction enzymes (Fermentas Life Sciences, Milan, Italy).

    other:

    Article Title: Quality control of overexpressed membrane proteins
    Article Snippet: Cultivation of E. coli MC1061 and E. coli TOP10 containing derivatives of pBADMycHisB (Invitrogen) was started with a 1–2% (vol/vol) inoculum of an overnight culture.

    Article Title: A New Family of Secreted Toxins in Pathogenic Neisseria Species
    Article Snippet: E. coli TOP10 (Life technologies) or BL21(DE3) (Life technologies) were grown at 37°C in liquid or solid Luria-Bertani (LB) medium (Difco), which contained appropriate antibiotics (50 µg/ml ticarcillin, 10 µg/ml chloramphenicol and/or 50 µg/ml kanamycin).

    Plasmid Preparation:

    Article Title: Balancing mcr-1 expression and bacterial survival is a delicate equilibrium between essential cellular defence mechanisms
    Article Snippet: .. In parallel standard curves for mcr-1 , 16S were obtained using as template serial dilutions of mcr-1- carring plasmid DNA extracted from pSU18-mcr-1 strain (4.3 pg of DNA corresponding to 106 copies, calculated through the website: http://cels.uri.edu/gsc/cndna.html ) and E. coli TOP10 (Invitrogen) total gDNA (5 ng corresponding to 106 cells) , respectively. .. Selection of induced high-level colistin-resistant mutants HLCRMs were generated from seven wild-type mcr-1 positive strains through 14-day serial passaging with increasing concentrations of colistin (Alfa Aesar, US).

    Article Title: Novel Bacterial Surface Display Systems Based on Outer Membrane Anchoring Elements from the Marine Bacterium Vibrio anguillarum ▿ ▿ †
    Article Snippet: .. E. coli Top10 [F− mcrA Δ( mrr-hsdRMS-mcrBC ) φ80 lacZ ΔM15 Δ lacX74 deoR recA1 araD139 Δ( ara-leu ) 7697 galU galK rpsL (Strr ) endA1 nupG ] (Invitrogen, Carlsbad, CA) was used as the host for recombinant plasmid construction and GFP surface display. ..

    Article Title: Transfer of KPC-2 Carbapenemase from Klebsiella pneumoniae to Escherichia coli in a Patient: First Case in Europe ▿
    Article Snippet: .. Purified plasmids from K. pneumoniae and E. coli were transformed into an E. coli Top10 (Invitrogen) recipient strain and selected on plates containing 100 mg/liter ampicillin; screening for bla KPC -positive colonies showed that, in both cases, E. coli Top10 was transformed with a 20-kb plasmid, which encoded KPC-2, bla TEM-1 , and bla OXA-9 , and not bla VIM-1 , which was located on a different plasmid. .. Plasmids from the transformed colonies were purified and digested with the HindIII and EcoRI restriction enzymes (Fermentas Life Sciences, Milan, Italy).

    Expressing:

    Article Title: Buwchitin: A Ruminal Peptide with Antimicrobial Potential against Enterococcus faecalis
    Article Snippet: .. E. coli TOP10 (Invitrogen, Carlsbad CA, USA) was used for cloning (to host expression vectors for protein expression). .. The pTrcHis TOPO® vector (Invitrogen, Carlsbad, CA, USA) was used to clone polymerase chain reaction (PCR) products for protein expression.

    Transformation Assay:

    Article Title: Transfer of KPC-2 Carbapenemase from Klebsiella pneumoniae to Escherichia coli in a Patient: First Case in Europe ▿
    Article Snippet: .. Purified plasmids from K. pneumoniae and E. coli were transformed into an E. coli Top10 (Invitrogen) recipient strain and selected on plates containing 100 mg/liter ampicillin; screening for bla KPC -positive colonies showed that, in both cases, E. coli Top10 was transformed with a 20-kb plasmid, which encoded KPC-2, bla TEM-1 , and bla OXA-9 , and not bla VIM-1 , which was located on a different plasmid. .. Plasmids from the transformed colonies were purified and digested with the HindIII and EcoRI restriction enzymes (Fermentas Life Sciences, Milan, Italy).

    Recombinant:

    Article Title: Genetic Determinants of Tetracycline Resistance in Vibrio harveyi
    Article Snippet: .. E. coli TOP10 (Invitrogen Corp., Carlsbad, Calif.) was used as the host strain for the recombinant plasmids. ..

    Article Title: Novel Bacterial Surface Display Systems Based on Outer Membrane Anchoring Elements from the Marine Bacterium Vibrio anguillarum ▿ ▿ †
    Article Snippet: .. E. coli Top10 [F− mcrA Δ( mrr-hsdRMS-mcrBC ) φ80 lacZ ΔM15 Δ lacX74 deoR recA1 araD139 Δ( ara-leu ) 7697 galU galK rpsL (Strr ) endA1 nupG ] (Invitrogen, Carlsbad, CA) was used as the host for recombinant plasmid construction and GFP surface display. ..

    Transmission Electron Microscopy:

    Article Title: Transfer of KPC-2 Carbapenemase from Klebsiella pneumoniae to Escherichia coli in a Patient: First Case in Europe ▿
    Article Snippet: .. Purified plasmids from K. pneumoniae and E. coli were transformed into an E. coli Top10 (Invitrogen) recipient strain and selected on plates containing 100 mg/liter ampicillin; screening for bla KPC -positive colonies showed that, in both cases, E. coli Top10 was transformed with a 20-kb plasmid, which encoded KPC-2, bla TEM-1 , and bla OXA-9 , and not bla VIM-1 , which was located on a different plasmid. .. Plasmids from the transformed colonies were purified and digested with the HindIII and EcoRI restriction enzymes (Fermentas Life Sciences, Milan, Italy).

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  • 89
    Thermo Fisher top10 escherichia coli
    DNA methylation in the enhancer region regulates perforin transcription. (a) Promoter region sequence containing enhancer in the PRF1 gene was aligned between human and mouse using the VISTA tool. The conservation degree is indicated by the peaks, with coloured peaks (blue for exon and light red for the non‐coding sequence) indicating > 70% conservation. Ten cytosine–phosphate–guanine sites (CpGs) in the enhancer are displayed upstream from the transcription start site (TSS). (b) Sorted CD8 + T cells from healthy donors were cultured initially in the presence of 200 IU/ml recombinant human interleukin (IL)‐2, 5 μg/ml plate‐coated anti‐CD3 and 1 μg/ml anti‐CD28 stimulating antibodies in vitro . 5‐Azacytidine (5‐aza) was added to the culture at a final concentration of 2·5 μM/ml for 48 h, followed by culture medium replacement and incubation for another 48 h. Flow cytometry data demonstrating perforin expression pre‐ and post‐treatment are shown. (c) Perforin – and perforin + CD8 + T cells were sorted. DNA was extracted and bisulfite‐converted from each cell subset, TA‐cloned to pCR4‐TOPO vector and transformed into <t>TOP10</t> <t>Escherichia</t> coli . Colonies were picked from each cell subset and their DNA was sequenced to measure DNA methylation status of the 10 CpGs in the enhancer. The data display rows represent individually sequenced clone. (d) The bar graphs display total DNA methylation percentage in each CpG from clones in (c).
    Top10 Escherichia Coli, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher e coli top10 carrying vector pcr4 topo
    Resistance against sulfonamide antibiotics mediated by SEW2_dhps01, SEW5_dhps01, AEW9_dhps01 , and AEG2_dhps01 . Five microliters of serially diluted E. coli <t>TOP10</t> cultures with starting OD 600 of 0.5 were spotted onto Iso-Sensitest agar plates supplemented with 1000 mg/L sulfamethazine (+ SMZ), 250 mg/L sulfamethoxazole (+ SMX), 250 mg/L sulfadiazine (+ SDZ) or 500 mg/L sulfisoxazole (+ SOX). Iso-Sensitest agar plates with no sulfonamide added (control) were also included. E. coli TOP10 cultures carrying the cloning vector <t>pCR4-TOPO,</t> pCR4_SEW2_dhps01, pCR4_SEW5_dhps01, pCR4_AEW9_dhps01 or pCR4_AEG2_dhps01 were considered.
    E Coli Top10 Carrying Vector Pcr4 Topo, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher escherichia coli
    Representative SDS-PAGE, 2-dimensional electrophoresis (2DE), and Western blots showing the workflow for selection of immunoreactive spots. (A) The 1-dimensional SDS-PAGE image of Staphylococcus aureus Newbould 305 trypsinized protein (lane 1) and <t>Escherichia</t> coli trypsinized protein (lane 2). (B) 2DE gel conducted on 7 cm, pH 4 to 7, immobilized pH gradient (IPG) strips of S. aureus Newbould 305 trypsinized protein. Mr = molecular weight. (C) Immunoblot of S. aureus Newbould 305 trypsinized protein using pooled bovine mastitic milk, with immunoreactive regions highlighted in red circles. (D) 2DE gel conducted on 7 cm, pH 4 to 7, IPG strips of Escherichia coli DH10-β trypsinized protein. (E) Immunoblot of E. coli DH10-β trypsinized protein using bovine mastitic milk, with the immunoreactive regions highlighted in red circles. Color version available online.
    Escherichia Coli, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 76 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    Thermo Fisher multishot flexplate top10 competent cells
    Analysis of the pop transcriptional unit. (A) Promoter activity assay for the promoter of pop , which was introduced in the promoterless GFP vector pProbe-NT. Mean fluorescence units of E. coli <t>Top10</t> cells with the different constructs in culture conditions as indicated are given. Error bars show standard deviations. Statistical significance between empty vector (grey bars) and the promoter construct (blue bars) and between growth conditions was tested with a Welch two sample t-test (**/++ p≤0.01; *** p≤0.001; ns, not significant). (B) Test for mono- or polycistronic mRNA. An agarose gel of RT-PCRs is shown. Two different forward primers, binding within ycbG (no. 23) or within pop (no. 24), were combined with a pop reverse primer (no. 25). L: 100 bp DNA Ladder (NEB); 23: PCR with primers 23+25; 24: PCR with primers 24+25. (C) Test for the predicted rho-independent terminator. An agarose gel of RT-PCRs is shown. Two different reverse primers, binding upstream (no. 27) or downstream (no. 28) of the stem loop structure, were combined with a pop forward primer (no. 26). L, 1 kb DNA Ladder (NEB); 27, PCR with primers 27+26; 28, PCR with primers 28+26; d. ORFs, downstream ORFs.
    Multishot Flexplate Top10 Competent Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    DNA methylation in the enhancer region regulates perforin transcription. (a) Promoter region sequence containing enhancer in the PRF1 gene was aligned between human and mouse using the VISTA tool. The conservation degree is indicated by the peaks, with coloured peaks (blue for exon and light red for the non‐coding sequence) indicating > 70% conservation. Ten cytosine–phosphate–guanine sites (CpGs) in the enhancer are displayed upstream from the transcription start site (TSS). (b) Sorted CD8 + T cells from healthy donors were cultured initially in the presence of 200 IU/ml recombinant human interleukin (IL)‐2, 5 μg/ml plate‐coated anti‐CD3 and 1 μg/ml anti‐CD28 stimulating antibodies in vitro . 5‐Azacytidine (5‐aza) was added to the culture at a final concentration of 2·5 μM/ml for 48 h, followed by culture medium replacement and incubation for another 48 h. Flow cytometry data demonstrating perforin expression pre‐ and post‐treatment are shown. (c) Perforin – and perforin + CD8 + T cells were sorted. DNA was extracted and bisulfite‐converted from each cell subset, TA‐cloned to pCR4‐TOPO vector and transformed into TOP10 Escherichia coli . Colonies were picked from each cell subset and their DNA was sequenced to measure DNA methylation status of the 10 CpGs in the enhancer. The data display rows represent individually sequenced clone. (d) The bar graphs display total DNA methylation percentage in each CpG from clones in (c).

    Journal: Clinical and Experimental Immunology

    Article Title: Tissue‐resident memory T cells are epigenetically cytotoxic with signs of exhaustion in human urinary bladder cancer

    doi: 10.1111/cei.13183

    Figure Lengend Snippet: DNA methylation in the enhancer region regulates perforin transcription. (a) Promoter region sequence containing enhancer in the PRF1 gene was aligned between human and mouse using the VISTA tool. The conservation degree is indicated by the peaks, with coloured peaks (blue for exon and light red for the non‐coding sequence) indicating > 70% conservation. Ten cytosine–phosphate–guanine sites (CpGs) in the enhancer are displayed upstream from the transcription start site (TSS). (b) Sorted CD8 + T cells from healthy donors were cultured initially in the presence of 200 IU/ml recombinant human interleukin (IL)‐2, 5 μg/ml plate‐coated anti‐CD3 and 1 μg/ml anti‐CD28 stimulating antibodies in vitro . 5‐Azacytidine (5‐aza) was added to the culture at a final concentration of 2·5 μM/ml for 48 h, followed by culture medium replacement and incubation for another 48 h. Flow cytometry data demonstrating perforin expression pre‐ and post‐treatment are shown. (c) Perforin – and perforin + CD8 + T cells were sorted. DNA was extracted and bisulfite‐converted from each cell subset, TA‐cloned to pCR4‐TOPO vector and transformed into TOP10 Escherichia coli . Colonies were picked from each cell subset and their DNA was sequenced to measure DNA methylation status of the 10 CpGs in the enhancer. The data display rows represent individually sequenced clone. (d) The bar graphs display total DNA methylation percentage in each CpG from clones in (c).

    Article Snippet: Next, the PCR amplicons were TA‐cloned into pCR4‐TOPO vector (ThermoFisher) and transformed into TOP10 Escherichia coli (ThermoFisher) by heat shock, according to the manufacturer’s protocol.

    Techniques: DNA Methylation Assay, Sequencing, Cell Culture, Recombinant, In Vitro, Concentration Assay, Incubation, Flow Cytometry, Cytometry, Expressing, Clone Assay, Plasmid Preparation, Transformation Assay

    Resistance against sulfonamide antibiotics mediated by SEW2_dhps01, SEW5_dhps01, AEW9_dhps01 , and AEG2_dhps01 . Five microliters of serially diluted E. coli TOP10 cultures with starting OD 600 of 0.5 were spotted onto Iso-Sensitest agar plates supplemented with 1000 mg/L sulfamethazine (+ SMZ), 250 mg/L sulfamethoxazole (+ SMX), 250 mg/L sulfadiazine (+ SDZ) or 500 mg/L sulfisoxazole (+ SOX). Iso-Sensitest agar plates with no sulfonamide added (control) were also included. E. coli TOP10 cultures carrying the cloning vector pCR4-TOPO, pCR4_SEW2_dhps01, pCR4_SEW5_dhps01, pCR4_AEW9_dhps01 or pCR4_AEG2_dhps01 were considered.

    Journal: Frontiers in Microbiology

    Article Title: Discovery of Novel Antibiotic Resistance Determinants in Forest and Grassland Soil Metagenomes

    doi: 10.3389/fmicb.2019.00460

    Figure Lengend Snippet: Resistance against sulfonamide antibiotics mediated by SEW2_dhps01, SEW5_dhps01, AEW9_dhps01 , and AEG2_dhps01 . Five microliters of serially diluted E. coli TOP10 cultures with starting OD 600 of 0.5 were spotted onto Iso-Sensitest agar plates supplemented with 1000 mg/L sulfamethazine (+ SMZ), 250 mg/L sulfamethoxazole (+ SMX), 250 mg/L sulfadiazine (+ SDZ) or 500 mg/L sulfisoxazole (+ SOX). Iso-Sensitest agar plates with no sulfonamide added (control) were also included. E. coli TOP10 cultures carrying the cloning vector pCR4-TOPO, pCR4_SEW2_dhps01, pCR4_SEW5_dhps01, pCR4_AEW9_dhps01 or pCR4_AEG2_dhps01 were considered.

    Article Snippet: E. coli TOP10 carrying vector pCR4-TOPO (Thermo Fisher Scientific) was used as control.

    Techniques: Clone Assay, Plasmid Preparation

    Antibiotic susceptibility profiles of E. coli TOP10 carrying soil-derived genes involved in antibiotic resistance. The genes were subcloned into plasmid vector pCR4-TOPO. MICs of antibiotics were determined using the broth microdilution method and are presented as fold increase relative to those for E. coli TOP10 carrying the cloning vector pCR4-TOPO. CAX, cefotaxime; CHL, chloramphenicol; ERY, erythromycin; GEN, gentamicin; LIN, lincomycin; RIF, rifampicin; SDZ, sulfadiazine; SMX, sulfamethoxazole; SMZ, sulfamethazine; SOX, sulfisoxazole; TET, tetracycline; TYL, tylosin.

    Journal: Frontiers in Microbiology

    Article Title: Discovery of Novel Antibiotic Resistance Determinants in Forest and Grassland Soil Metagenomes

    doi: 10.3389/fmicb.2019.00460

    Figure Lengend Snippet: Antibiotic susceptibility profiles of E. coli TOP10 carrying soil-derived genes involved in antibiotic resistance. The genes were subcloned into plasmid vector pCR4-TOPO. MICs of antibiotics were determined using the broth microdilution method and are presented as fold increase relative to those for E. coli TOP10 carrying the cloning vector pCR4-TOPO. CAX, cefotaxime; CHL, chloramphenicol; ERY, erythromycin; GEN, gentamicin; LIN, lincomycin; RIF, rifampicin; SDZ, sulfadiazine; SMX, sulfamethoxazole; SMZ, sulfamethazine; SOX, sulfisoxazole; TET, tetracycline; TYL, tylosin.

    Article Snippet: E. coli TOP10 carrying vector pCR4-TOPO (Thermo Fisher Scientific) was used as control.

    Techniques: Derivative Assay, Plasmid Preparation, Clone Assay

    Representative SDS-PAGE, 2-dimensional electrophoresis (2DE), and Western blots showing the workflow for selection of immunoreactive spots. (A) The 1-dimensional SDS-PAGE image of Staphylococcus aureus Newbould 305 trypsinized protein (lane 1) and Escherichia coli trypsinized protein (lane 2). (B) 2DE gel conducted on 7 cm, pH 4 to 7, immobilized pH gradient (IPG) strips of S. aureus Newbould 305 trypsinized protein. Mr = molecular weight. (C) Immunoblot of S. aureus Newbould 305 trypsinized protein using pooled bovine mastitic milk, with immunoreactive regions highlighted in red circles. (D) 2DE gel conducted on 7 cm, pH 4 to 7, IPG strips of Escherichia coli DH10-β trypsinized protein. (E) Immunoblot of E. coli DH10-β trypsinized protein using bovine mastitic milk, with the immunoreactive regions highlighted in red circles. Color version available online.

    Journal: Journal of dairy science

    Article Title: Immunoproteomics to identify Staphylococcus aureus antigens expressed in bovine milk during mastitis

    doi: 10.3168/jds.2017-14040

    Figure Lengend Snippet: Representative SDS-PAGE, 2-dimensional electrophoresis (2DE), and Western blots showing the workflow for selection of immunoreactive spots. (A) The 1-dimensional SDS-PAGE image of Staphylococcus aureus Newbould 305 trypsinized protein (lane 1) and Escherichia coli trypsinized protein (lane 2). (B) 2DE gel conducted on 7 cm, pH 4 to 7, immobilized pH gradient (IPG) strips of S. aureus Newbould 305 trypsinized protein. Mr = molecular weight. (C) Immunoblot of S. aureus Newbould 305 trypsinized protein using pooled bovine mastitic milk, with immunoreactive regions highlighted in red circles. (D) 2DE gel conducted on 7 cm, pH 4 to 7, IPG strips of Escherichia coli DH10-β trypsinized protein. (E) Immunoblot of E. coli DH10-β trypsinized protein using bovine mastitic milk, with the immunoreactive regions highlighted in red circles. Color version available online.

    Article Snippet: Briefly, S. aureus Newbould 305, S. aureus C1, and Escherichia coli (DH10-β Top10, Thermo Fisher Scientific, Waltham, MA) were grown in LIM overnight to an optical density ( OD ) of 0.75 to 1.2 and harvested by centrifugation (6,000 × g for 10 min at 4°C), before washing 3 times with 1× PBS.

    Techniques: SDS Page, Electrophoresis, Two-Dimensional Gel Electrophoresis, Western Blot, Selection, Molecular Weight

    Analysis of the pop transcriptional unit. (A) Promoter activity assay for the promoter of pop , which was introduced in the promoterless GFP vector pProbe-NT. Mean fluorescence units of E. coli Top10 cells with the different constructs in culture conditions as indicated are given. Error bars show standard deviations. Statistical significance between empty vector (grey bars) and the promoter construct (blue bars) and between growth conditions was tested with a Welch two sample t-test (**/++ p≤0.01; *** p≤0.001; ns, not significant). (B) Test for mono- or polycistronic mRNA. An agarose gel of RT-PCRs is shown. Two different forward primers, binding within ycbG (no. 23) or within pop (no. 24), were combined with a pop reverse primer (no. 25). L: 100 bp DNA Ladder (NEB); 23: PCR with primers 23+25; 24: PCR with primers 24+25. (C) Test for the predicted rho-independent terminator. An agarose gel of RT-PCRs is shown. Two different reverse primers, binding upstream (no. 27) or downstream (no. 28) of the stem loop structure, were combined with a pop forward primer (no. 26). L, 1 kb DNA Ladder (NEB); 27, PCR with primers 27+26; 28, PCR with primers 28+26; d. ORFs, downstream ORFs.

    Journal: bioRxiv

    Article Title: A novel pH-regulated, unusual 603 bp overlapping protein coding gene pop is encoded antisense to ompA in Escherichia coli O157:H7 (EHEC)

    doi: 10.1101/852251

    Figure Lengend Snippet: Analysis of the pop transcriptional unit. (A) Promoter activity assay for the promoter of pop , which was introduced in the promoterless GFP vector pProbe-NT. Mean fluorescence units of E. coli Top10 cells with the different constructs in culture conditions as indicated are given. Error bars show standard deviations. Statistical significance between empty vector (grey bars) and the promoter construct (blue bars) and between growth conditions was tested with a Welch two sample t-test (**/++ p≤0.01; *** p≤0.001; ns, not significant). (B) Test for mono- or polycistronic mRNA. An agarose gel of RT-PCRs is shown. Two different forward primers, binding within ycbG (no. 23) or within pop (no. 24), were combined with a pop reverse primer (no. 25). L: 100 bp DNA Ladder (NEB); 23: PCR with primers 23+25; 24: PCR with primers 24+25. (C) Test for the predicted rho-independent terminator. An agarose gel of RT-PCRs is shown. Two different reverse primers, binding upstream (no. 27) or downstream (no. 28) of the stem loop structure, were combined with a pop forward primer (no. 26). L, 1 kb DNA Ladder (NEB); 27, PCR with primers 27+26; 28, PCR with primers 28+26; d. ORFs, downstream ORFs.

    Article Snippet: Vector constructs were transformed in E. coli Top10 cells and plated on LB with required antibiotics.

    Techniques: Activity Assay, Plasmid Preparation, Fluorescence, Construct, Agarose Gel Electrophoresis, Binding Assay, Polymerase Chain Reaction