escherichia coli strains Thermo Fisher Search Results


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  • 90
    Thermo Fisher one shot stbl3 chemically competent e coli
    One Shot Stbl3 Chemically Competent E Coli, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 294 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    77
    Thermo Fisher escherichia coli strains e coli strain dh5α
    ΦTE-F expresses ToxI RNA during infection. (A) Upper panel; an S1-nuclease protection assay was used to detect ToxI levels from a ToxIN plasmid during ΦTE wt infection, using an antisense probe against the full 5.5 repeat ToxI sequence [46] . The antisense ToxI-probe was first hybridised to 10 µg of total RNA prepared from Pba ToxIN (pMJ4) at different times after ΦTE infection, and then followed by S1-nuclease treatment. Numbers (+) indicate the time (min) after infection or (−) without the addition of phage. Pba ToxIN-FS (pTA47) and Pba serve as positive and negative controls, respectively. A non-hybridized S1-digested probe (+S1) serves as a further negative control. <t>DH5α</t> 1.5 repeats (pTA96), a non-S1 digested probe (−S1) and an in vitro transcribed Hammerhead ribozyme (HHRz), which cleaves itself during transcription, serve as size markers. HHRz was prepared as described previously [45] . Lower panel; Western blot targeting C-terminal FLAG tagged ToxN contained within total protein harvested from Pba ToxIN (pMJ4) at different time points, with (+, left) and without (−, right) phage infection. Time 0 indicates a sample taken immediately after infection. Total protein from Pba ToxIN (pMJ4) (−) serves as positive control. (B) Infection with escape phage ΦTE-F. Levels of ToxI were determined by S1-assay (upper) as described in (A) with and without infection. ToxN levels were estimated by Western blotting (lower) as described in (A). (C) Expression of the ΦTE-F ToxI locus. An S1-nuclease assay targeting ToxI was performed on total RNA of Pba (pBR322) at different times during ΦTE-F infection. Pba ToxIN (pMJ4) and DH5α serve as positive and negative controls, respectively.
    Escherichia Coli Strains E Coli Strain Dh5α, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 77/100, based on 182 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Thermo Fisher escherichia coli strain top10f
    MotB is toxic to E. coli. Heterologous expression of motB in ( a ) BL21(DE3) or ( b ) <t>TOP10F’</t> E. coli causes cell lysis or inhibits growth, respectively. Cell lines containing either the empty vector pNW129 (circles) or expression vector pNW129-MotB (squares) were induced with 0.2% ( w / v ) arabinose (final concentration) at an OD 600 between 0.3 and 0.4. Growth curves are representative of three independent replicates. To monitor the amount of MotB produced, cell aliquots at 0, 60 and 90 min post-induction were analyzed by SDS-PAGE. The protein gel slice containing MotB is shown for a representative experiment with the band corresponding to MotB designated by a black arrow. It should be noted that although MotB production is seen at 60 min for BL21(DE3)/pNW129-MotB, no additional accumulation is apparent at 90 min perhaps because of the significant cell death at this time point.
    Escherichia Coli Strain Top10f, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 219 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    Thermo Fisher escherichia coli strain mach1
    MotB is toxic to E. coli. Heterologous expression of motB in ( a ) BL21(DE3) or ( b ) <t>TOP10F’</t> E. coli causes cell lysis or inhibits growth, respectively. Cell lines containing either the empty vector pNW129 (circles) or expression vector pNW129-MotB (squares) were induced with 0.2% ( w / v ) arabinose (final concentration) at an OD 600 between 0.3 and 0.4. Growth curves are representative of three independent replicates. To monitor the amount of MotB produced, cell aliquots at 0, 60 and 90 min post-induction were analyzed by SDS-PAGE. The protein gel slice containing MotB is shown for a representative experiment with the band corresponding to MotB designated by a black arrow. It should be noted that although MotB production is seen at 60 min for BL21(DE3)/pNW129-MotB, no additional accumulation is apparent at 90 min perhaps because of the significant cell death at this time point.
    Escherichia Coli Strain Mach1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 79/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    77
    Thermo Fisher escherichia coli strain top1o
    MotB is toxic to E. coli. Heterologous expression of motB in ( a ) BL21(DE3) or ( b ) <t>TOP10F’</t> E. coli causes cell lysis or inhibits growth, respectively. Cell lines containing either the empty vector pNW129 (circles) or expression vector pNW129-MotB (squares) were induced with 0.2% ( w / v ) arabinose (final concentration) at an OD 600 between 0.3 and 0.4. Growth curves are representative of three independent replicates. To monitor the amount of MotB produced, cell aliquots at 0, 60 and 90 min post-induction were analyzed by SDS-PAGE. The protein gel slice containing MotB is shown for a representative experiment with the band corresponding to MotB designated by a black arrow. It should be noted that although MotB production is seen at 60 min for BL21(DE3)/pNW129-MotB, no additional accumulation is apparent at 90 min perhaps because of the significant cell death at this time point.
    Escherichia Coli Strain Top1o, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 77/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher escherichia coli strains top10
    Barrier RFB1 maps to a 78-bp DNA fragment that is specifically recognized by a binding protein. (A) Diagram of the nontranscribed spacer of the S. pombe rRNA gene, with the locations of the three barriers (RFB1-3) and the replication origin ars3001 indicated. Below the map, lines a to f correspond to the deleted fragments inserted in pIRT2 and assayed as shown in panel C. The names of the resultant plasmids and the results obtained are shown to the right. (B) Map of vector pIRT2 indicating the site where the fragments shown in panel A were cloned (insert). (C) RFB1 activity of fragments a to f in wild-type strain 35 (panels a to f) and RFB1 activity of fragment e in swi1 (EN3182) and swi3 (EN3366) mutant strains (panels e- swi1 and e- swi3 ). Arrows point to the signals corresponding to Y-shaped accumulated replication intermediates with the fork arrested at RFB1. (D) EMSA using labeled fragment e and the indicated amounts of protein extract. In lane 6, a 166-fold excess of unlabeled fragment e was added to the binding reaction. (E) Expression of Sap1p in E. coli <t>TOP10</t> cells was induced by the addition of 0.02% arabinose to the culture for 2 h. Proteins from 2.5 × 10 8 cells were separated in 12% SDS-polyacrylamide gels (lanes 1 and 2) and transferred to polyvinylidene difluoride membranes, and His 6 -Sap1p was detected with anti-His 6 -peroxidase antibody (Roche) (lanes 3 and 4). Lanes 5 and 6 correspond to mobility shift assays using 4.7 μg of a protein extract from noninduced (−) or induced (+) cells and the same fragment as that used for panel D.
    Escherichia Coli Strains Top10, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 296 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    80
    Thermo Fisher escherichia coli strain rosetta
    Barrier RFB1 maps to a 78-bp DNA fragment that is specifically recognized by a binding protein. (A) Diagram of the nontranscribed spacer of the S. pombe rRNA gene, with the locations of the three barriers (RFB1-3) and the replication origin ars3001 indicated. Below the map, lines a to f correspond to the deleted fragments inserted in pIRT2 and assayed as shown in panel C. The names of the resultant plasmids and the results obtained are shown to the right. (B) Map of vector pIRT2 indicating the site where the fragments shown in panel A were cloned (insert). (C) RFB1 activity of fragments a to f in wild-type strain 35 (panels a to f) and RFB1 activity of fragment e in swi1 (EN3182) and swi3 (EN3366) mutant strains (panels e- swi1 and e- swi3 ). Arrows point to the signals corresponding to Y-shaped accumulated replication intermediates with the fork arrested at RFB1. (D) EMSA using labeled fragment e and the indicated amounts of protein extract. In lane 6, a 166-fold excess of unlabeled fragment e was added to the binding reaction. (E) Expression of Sap1p in E. coli <t>TOP10</t> cells was induced by the addition of 0.02% arabinose to the culture for 2 h. Proteins from 2.5 × 10 8 cells were separated in 12% SDS-polyacrylamide gels (lanes 1 and 2) and transferred to polyvinylidene difluoride membranes, and His 6 -Sap1p was detected with anti-His 6 -peroxidase antibody (Roche) (lanes 3 and 4). Lanes 5 and 6 correspond to mobility shift assays using 4.7 μg of a protein extract from noninduced (−) or induced (+) cells and the same fragment as that used for panel D.
    Escherichia Coli Strain Rosetta, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 80/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Thermo Fisher escherichia coli strain dh5αmcr
    Barrier RFB1 maps to a 78-bp DNA fragment that is specifically recognized by a binding protein. (A) Diagram of the nontranscribed spacer of the S. pombe rRNA gene, with the locations of the three barriers (RFB1-3) and the replication origin ars3001 indicated. Below the map, lines a to f correspond to the deleted fragments inserted in pIRT2 and assayed as shown in panel C. The names of the resultant plasmids and the results obtained are shown to the right. (B) Map of vector pIRT2 indicating the site where the fragments shown in panel A were cloned (insert). (C) RFB1 activity of fragments a to f in wild-type strain 35 (panels a to f) and RFB1 activity of fragment e in swi1 (EN3182) and swi3 (EN3366) mutant strains (panels e- swi1 and e- swi3 ). Arrows point to the signals corresponding to Y-shaped accumulated replication intermediates with the fork arrested at RFB1. (D) EMSA using labeled fragment e and the indicated amounts of protein extract. In lane 6, a 166-fold excess of unlabeled fragment e was added to the binding reaction. (E) Expression of Sap1p in E. coli <t>TOP10</t> cells was induced by the addition of 0.02% arabinose to the culture for 2 h. Proteins from 2.5 × 10 8 cells were separated in 12% SDS-polyacrylamide gels (lanes 1 and 2) and transferred to polyvinylidene difluoride membranes, and His 6 -Sap1p was detected with anti-His 6 -peroxidase antibody (Roche) (lanes 3 and 4). Lanes 5 and 6 correspond to mobility shift assays using 4.7 μg of a protein extract from noninduced (−) or induced (+) cells and the same fragment as that used for panel D.
    Escherichia Coli Strain Dh5αmcr, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Thermo Fisher escherichia coli strain bl21
    Protein analysis of pET-SUMO-E2-T1 expression in E. coli <t>BL21</t> (DE3) . (A) Protein from soluble and insoluble fractions separated by electrophoresis on 10% Bis-Tris gel and stained with Coomassie blue. Lane 1, SeeBlue ® Plus2 MW standard; lane 2, soluble protein fraction at 0 hours; lane 3, insoluble protein fraction at 0 hours; lane 4, soluble protein fraction 2 hours post IPTG induction; lane 5, insoluble protein fraction 2 hours IPTG post induction; lane 6, E2-T1 purified from IB (0.4 μL); lane 7, E2-T1 purified from IB (2 μL). (B) Western blot image of E2-T1 protein expressed in E. coli BL21 (DE3). Equivalent amounts of protein from the soluble and insoluble fractions were transferred to Hybond C and incubated with an anti-his antibody and detected by ECL. Lane 1, soluble protein fraction 0 hours; lane 2, insoluble protein fraction 0 hours; lane 3, soluble protein fraction 2 hours post IPTG induction; lane 4, insoluble protein fraction 2 hours post IPTG induction.
    Escherichia Coli Strain Bl21, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 942 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    87
    Thermo Fisher escherichia coli strain dh12s
    Protein analysis of pET-SUMO-E2-T1 expression in E. coli <t>BL21</t> (DE3) . (A) Protein from soluble and insoluble fractions separated by electrophoresis on 10% Bis-Tris gel and stained with Coomassie blue. Lane 1, SeeBlue ® Plus2 MW standard; lane 2, soluble protein fraction at 0 hours; lane 3, insoluble protein fraction at 0 hours; lane 4, soluble protein fraction 2 hours post IPTG induction; lane 5, insoluble protein fraction 2 hours IPTG post induction; lane 6, E2-T1 purified from IB (0.4 μL); lane 7, E2-T1 purified from IB (2 μL). (B) Western blot image of E2-T1 protein expressed in E. coli BL21 (DE3). Equivalent amounts of protein from the soluble and insoluble fractions were transferred to Hybond C and incubated with an anti-his antibody and detected by ECL. Lane 1, soluble protein fraction 0 hours; lane 2, insoluble protein fraction 0 hours; lane 3, soluble protein fraction 2 hours post IPTG induction; lane 4, insoluble protein fraction 2 hours post IPTG induction.
    Escherichia Coli Strain Dh12s, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 87/100, based on 42 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher escherichia coli strain dh10b
    Strategy for constructing a self-excisable RhCMV BAC. (A) Cloning of the full-length RhCMV genome into a BAC vector by Cre/lox recombination. The RhCMV genome structure with expansion of the US1-to-US3 region of recombinant viruses is diagrammed. The open box represents the internal junction between L and S components of the RhCMV genome. Cre/lox recombination was performed in Telo-RF cells. Recombinant clones containing the BAC vector in the viral genome (vRhCMV/BAC-EGFP) were plaque purified, and circular-form viral DNA was transformed into E. coli strain <t>DH10B</t> for plasmid isolation. (B) Substitution of the cre ORF for the egfp ORF by allelic exchange in E. coli . Only a portion of each plasmid is illustrated. The homologous flanking regions and the cre ORF of the recombination vector, pWC212, are illustrated. UL, unique sequences of the L component; US, unique sequences of the S component; PSV40, SV40 promoter; poly A, SV40 polyadenylation signal.
    Escherichia Coli Strain Dh10b, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 237 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    Thermo Fisher escherichia coli strain machi
    Strategy for constructing a self-excisable RhCMV BAC. (A) Cloning of the full-length RhCMV genome into a BAC vector by Cre/lox recombination. The RhCMV genome structure with expansion of the US1-to-US3 region of recombinant viruses is diagrammed. The open box represents the internal junction between L and S components of the RhCMV genome. Cre/lox recombination was performed in Telo-RF cells. Recombinant clones containing the BAC vector in the viral genome (vRhCMV/BAC-EGFP) were plaque purified, and circular-form viral DNA was transformed into E. coli strain <t>DH10B</t> for plasmid isolation. (B) Substitution of the cre ORF for the egfp ORF by allelic exchange in E. coli . Only a portion of each plasmid is illustrated. The homologous flanking regions and the cre ORF of the recombination vector, pWC212, are illustrated. UL, unique sequences of the L component; US, unique sequences of the S component; PSV40, SV40 promoter; poly A, SV40 polyadenylation signal.
    Escherichia Coli Strain Machi, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 84/100, based on 30 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher escherichia coli strain genehogs
    Strategy for constructing a self-excisable RhCMV BAC. (A) Cloning of the full-length RhCMV genome into a BAC vector by Cre/lox recombination. The RhCMV genome structure with expansion of the US1-to-US3 region of recombinant viruses is diagrammed. The open box represents the internal junction between L and S components of the RhCMV genome. Cre/lox recombination was performed in Telo-RF cells. Recombinant clones containing the BAC vector in the viral genome (vRhCMV/BAC-EGFP) were plaque purified, and circular-form viral DNA was transformed into E. coli strain <t>DH10B</t> for plasmid isolation. (B) Substitution of the cre ORF for the egfp ORF by allelic exchange in E. coli . Only a portion of each plasmid is illustrated. The homologous flanking regions and the cre ORF of the recombination vector, pWC212, are illustrated. UL, unique sequences of the L component; US, unique sequences of the S component; PSV40, SV40 promoter; poly A, SV40 polyadenylation signal.
    Escherichia Coli Strain Genehogs, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 87/100, based on 113 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Thermo Fisher escherichia coli strain dh10bac
    Strategy for constructing a self-excisable RhCMV BAC. (A) Cloning of the full-length RhCMV genome into a BAC vector by Cre/lox recombination. The RhCMV genome structure with expansion of the US1-to-US3 region of recombinant viruses is diagrammed. The open box represents the internal junction between L and S components of the RhCMV genome. Cre/lox recombination was performed in Telo-RF cells. Recombinant clones containing the BAC vector in the viral genome (vRhCMV/BAC-EGFP) were plaque purified, and circular-form viral DNA was transformed into E. coli strain <t>DH10B</t> for plasmid isolation. (B) Substitution of the cre ORF for the egfp ORF by allelic exchange in E. coli . Only a portion of each plasmid is illustrated. The homologous flanking regions and the cre ORF of the recombination vector, pWC212, are illustrated. UL, unique sequences of the L component; US, unique sequences of the S component; PSV40, SV40 promoter; poly A, SV40 polyadenylation signal.
    Escherichia Coli Strain Dh10bac, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 172 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    Thermo Fisher escherichia coli strain topxf
    Strategy for constructing a self-excisable RhCMV BAC. (A) Cloning of the full-length RhCMV genome into a BAC vector by Cre/lox recombination. The RhCMV genome structure with expansion of the US1-to-US3 region of recombinant viruses is diagrammed. The open box represents the internal junction between L and S components of the RhCMV genome. Cre/lox recombination was performed in Telo-RF cells. Recombinant clones containing the BAC vector in the viral genome (vRhCMV/BAC-EGFP) were plaque purified, and circular-form viral DNA was transformed into E. coli strain <t>DH10B</t> for plasmid isolation. (B) Substitution of the cre ORF for the egfp ORF by allelic exchange in E. coli . Only a portion of each plasmid is illustrated. The homologous flanking regions and the cre ORF of the recombination vector, pWC212, are illustrated. UL, unique sequences of the L component; US, unique sequences of the S component; PSV40, SV40 promoter; poly A, SV40 polyadenylation signal.
    Escherichia Coli Strain Topxf, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 79/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    83
    Thermo Fisher escherichia coli strain dh10β
    Strategy for constructing a self-excisable RhCMV BAC. (A) Cloning of the full-length RhCMV genome into a BAC vector by Cre/lox recombination. The RhCMV genome structure with expansion of the US1-to-US3 region of recombinant viruses is diagrammed. The open box represents the internal junction between L and S components of the RhCMV genome. Cre/lox recombination was performed in Telo-RF cells. Recombinant clones containing the BAC vector in the viral genome (vRhCMV/BAC-EGFP) were plaque purified, and circular-form viral DNA was transformed into E. coli strain <t>DH10B</t> for plasmid isolation. (B) Substitution of the cre ORF for the egfp ORF by allelic exchange in E. coli . Only a portion of each plasmid is illustrated. The homologous flanking regions and the cre ORF of the recombination vector, pWC212, are illustrated. UL, unique sequences of the L component; US, unique sequences of the S component; PSV40, SV40 promoter; poly A, SV40 polyadenylation signal.
    Escherichia Coli Strain Dh10β, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 83/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher escherichia coli strains stbl3
    Strategy for constructing a self-excisable RhCMV BAC. (A) Cloning of the full-length RhCMV genome into a BAC vector by Cre/lox recombination. The RhCMV genome structure with expansion of the US1-to-US3 region of recombinant viruses is diagrammed. The open box represents the internal junction between L and S components of the RhCMV genome. Cre/lox recombination was performed in Telo-RF cells. Recombinant clones containing the BAC vector in the viral genome (vRhCMV/BAC-EGFP) were plaque purified, and circular-form viral DNA was transformed into E. coli strain <t>DH10B</t> for plasmid isolation. (B) Substitution of the cre ORF for the egfp ORF by allelic exchange in E. coli . Only a portion of each plasmid is illustrated. The homologous flanking regions and the cre ORF of the recombination vector, pWC212, are illustrated. UL, unique sequences of the L component; US, unique sequences of the S component; PSV40, SV40 promoter; poly A, SV40 polyadenylation signal.
    Escherichia Coli Strains Stbl3, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 77/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    81
    Thermo Fisher escherichia coli strain stbl2
    Strategy for constructing a self-excisable RhCMV BAC. (A) Cloning of the full-length RhCMV genome into a BAC vector by Cre/lox recombination. The RhCMV genome structure with expansion of the US1-to-US3 region of recombinant viruses is diagrammed. The open box represents the internal junction between L and S components of the RhCMV genome. Cre/lox recombination was performed in Telo-RF cells. Recombinant clones containing the BAC vector in the viral genome (vRhCMV/BAC-EGFP) were plaque purified, and circular-form viral DNA was transformed into E. coli strain <t>DH10B</t> for plasmid isolation. (B) Substitution of the cre ORF for the egfp ORF by allelic exchange in E. coli . Only a portion of each plasmid is illustrated. The homologous flanking regions and the cre ORF of the recombination vector, pWC212, are illustrated. UL, unique sequences of the L component; US, unique sequences of the S component; PSV40, SV40 promoter; poly A, SV40 polyadenylation signal.
    Escherichia Coli Strain Stbl2, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 81/100, based on 55 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher escherichia coli strain bl21de3
    Strategy for constructing a self-excisable RhCMV BAC. (A) Cloning of the full-length RhCMV genome into a BAC vector by Cre/lox recombination. The RhCMV genome structure with expansion of the US1-to-US3 region of recombinant viruses is diagrammed. The open box represents the internal junction between L and S components of the RhCMV genome. Cre/lox recombination was performed in Telo-RF cells. Recombinant clones containing the BAC vector in the viral genome (vRhCMV/BAC-EGFP) were plaque purified, and circular-form viral DNA was transformed into E. coli strain <t>DH10B</t> for plasmid isolation. (B) Substitution of the cre ORF for the egfp ORF by allelic exchange in E. coli . Only a portion of each plasmid is illustrated. The homologous flanking regions and the cre ORF of the recombination vector, pWC212, are illustrated. UL, unique sequences of the L component; US, unique sequences of the S component; PSV40, SV40 promoter; poly A, SV40 polyadenylation signal.
    Escherichia Coli Strain Bl21de3, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 82/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher escherichia coli strain dh5a
    Strategy for constructing a self-excisable RhCMV BAC. (A) Cloning of the full-length RhCMV genome into a BAC vector by Cre/lox recombination. The RhCMV genome structure with expansion of the US1-to-US3 region of recombinant viruses is diagrammed. The open box represents the internal junction between L and S components of the RhCMV genome. Cre/lox recombination was performed in Telo-RF cells. Recombinant clones containing the BAC vector in the viral genome (vRhCMV/BAC-EGFP) were plaque purified, and circular-form viral DNA was transformed into E. coli strain <t>DH10B</t> for plasmid isolation. (B) Substitution of the cre ORF for the egfp ORF by allelic exchange in E. coli . Only a portion of each plasmid is illustrated. The homologous flanking regions and the cre ORF of the recombination vector, pWC212, are illustrated. UL, unique sequences of the L component; US, unique sequences of the S component; PSV40, SV40 promoter; poly A, SV40 polyadenylation signal.
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    Thermo Fisher escherichia coli strains jm109
    Strategy for constructing a self-excisable RhCMV BAC. (A) Cloning of the full-length RhCMV genome into a BAC vector by Cre/lox recombination. The RhCMV genome structure with expansion of the US1-to-US3 region of recombinant viruses is diagrammed. The open box represents the internal junction between L and S components of the RhCMV genome. Cre/lox recombination was performed in Telo-RF cells. Recombinant clones containing the BAC vector in the viral genome (vRhCMV/BAC-EGFP) were plaque purified, and circular-form viral DNA was transformed into E. coli strain <t>DH10B</t> for plasmid isolation. (B) Substitution of the cre ORF for the egfp ORF by allelic exchange in E. coli . Only a portion of each plasmid is illustrated. The homologous flanking regions and the cre ORF of the recombination vector, pWC212, are illustrated. UL, unique sequences of the L component; US, unique sequences of the S component; PSV40, SV40 promoter; poly A, SV40 polyadenylation signal.
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    Thermo Fisher competent escherichia coli strain
    Strategy for constructing a self-excisable RhCMV BAC. (A) Cloning of the full-length RhCMV genome into a BAC vector by Cre/lox recombination. The RhCMV genome structure with expansion of the US1-to-US3 region of recombinant viruses is diagrammed. The open box represents the internal junction between L and S components of the RhCMV genome. Cre/lox recombination was performed in Telo-RF cells. Recombinant clones containing the BAC vector in the viral genome (vRhCMV/BAC-EGFP) were plaque purified, and circular-form viral DNA was transformed into E. coli strain <t>DH10B</t> for plasmid isolation. (B) Substitution of the cre ORF for the egfp ORF by allelic exchange in E. coli . Only a portion of each plasmid is illustrated. The homologous flanking regions and the cre ORF of the recombination vector, pWC212, are illustrated. UL, unique sequences of the L component; US, unique sequences of the S component; PSV40, SV40 promoter; poly A, SV40 polyadenylation signal.
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    Thermo Fisher host escherichia coli strain
    Strategy for constructing a self-excisable RhCMV BAC. (A) Cloning of the full-length RhCMV genome into a BAC vector by Cre/lox recombination. The RhCMV genome structure with expansion of the US1-to-US3 region of recombinant viruses is diagrammed. The open box represents the internal junction between L and S components of the RhCMV genome. Cre/lox recombination was performed in Telo-RF cells. Recombinant clones containing the BAC vector in the viral genome (vRhCMV/BAC-EGFP) were plaque purified, and circular-form viral DNA was transformed into E. coli strain <t>DH10B</t> for plasmid isolation. (B) Substitution of the cre ORF for the egfp ORF by allelic exchange in E. coli . Only a portion of each plasmid is illustrated. The homologous flanking regions and the cre ORF of the recombination vector, pWC212, are illustrated. UL, unique sequences of the L component; US, unique sequences of the S component; PSV40, SV40 promoter; poly A, SV40 polyadenylation signal.
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    Thermo Fisher escherichia coli strain bl21star
    Strategy for constructing a self-excisable RhCMV BAC. (A) Cloning of the full-length RhCMV genome into a BAC vector by Cre/lox recombination. The RhCMV genome structure with expansion of the US1-to-US3 region of recombinant viruses is diagrammed. The open box represents the internal junction between L and S components of the RhCMV genome. Cre/lox recombination was performed in Telo-RF cells. Recombinant clones containing the BAC vector in the viral genome (vRhCMV/BAC-EGFP) were plaque purified, and circular-form viral DNA was transformed into E. coli strain <t>DH10B</t> for plasmid isolation. (B) Substitution of the cre ORF for the egfp ORF by allelic exchange in E. coli . Only a portion of each plasmid is illustrated. The homologous flanking regions and the cre ORF of the recombination vector, pWC212, are illustrated. UL, unique sequences of the L component; US, unique sequences of the S component; PSV40, SV40 promoter; poly A, SV40 polyadenylation signal.
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    Thermo Fisher escherichia coli strain dh5
    Strategy for constructing a self-excisable RhCMV BAC. (A) Cloning of the full-length RhCMV genome into a BAC vector by Cre/lox recombination. The RhCMV genome structure with expansion of the US1-to-US3 region of recombinant viruses is diagrammed. The open box represents the internal junction between L and S components of the RhCMV genome. Cre/lox recombination was performed in Telo-RF cells. Recombinant clones containing the BAC vector in the viral genome (vRhCMV/BAC-EGFP) were plaque purified, and circular-form viral DNA was transformed into E. coli strain <t>DH10B</t> for plasmid isolation. (B) Substitution of the cre ORF for the egfp ORF by allelic exchange in E. coli . Only a portion of each plasmid is illustrated. The homologous flanking regions and the cre ORF of the recombination vector, pWC212, are illustrated. UL, unique sequences of the L component; US, unique sequences of the S component; PSV40, SV40 promoter; poly A, SV40 polyadenylation signal.
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    Thermo Fisher escherichia coli strain invαf
    Strategy for constructing a self-excisable RhCMV BAC. (A) Cloning of the full-length RhCMV genome into a BAC vector by Cre/lox recombination. The RhCMV genome structure with expansion of the US1-to-US3 region of recombinant viruses is diagrammed. The open box represents the internal junction between L and S components of the RhCMV genome. Cre/lox recombination was performed in Telo-RF cells. Recombinant clones containing the BAC vector in the viral genome (vRhCMV/BAC-EGFP) were plaque purified, and circular-form viral DNA was transformed into E. coli strain <t>DH10B</t> for plasmid isolation. (B) Substitution of the cre ORF for the egfp ORF by allelic exchange in E. coli . Only a portion of each plasmid is illustrated. The homologous flanking regions and the cre ORF of the recombination vector, pWC212, are illustrated. UL, unique sequences of the L component; US, unique sequences of the S component; PSV40, SV40 promoter; poly A, SV40 polyadenylation signal.
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    Thermo Fisher escherichia coli strain dh5amcrab
    Strategy for constructing a self-excisable RhCMV BAC. (A) Cloning of the full-length RhCMV genome into a BAC vector by Cre/lox recombination. The RhCMV genome structure with expansion of the US1-to-US3 region of recombinant viruses is diagrammed. The open box represents the internal junction between L and S components of the RhCMV genome. Cre/lox recombination was performed in Telo-RF cells. Recombinant clones containing the BAC vector in the viral genome (vRhCMV/BAC-EGFP) were plaque purified, and circular-form viral DNA was transformed into E. coli strain <t>DH10B</t> for plasmid isolation. (B) Substitution of the cre ORF for the egfp ORF by allelic exchange in E. coli . Only a portion of each plasmid is illustrated. The homologous flanking regions and the cre ORF of the recombination vector, pWC212, are illustrated. UL, unique sequences of the L component; US, unique sequences of the S component; PSV40, SV40 promoter; poly A, SV40 polyadenylation signal.
    Escherichia Coli Strain Dh5amcrab, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 79/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher escherichia coli er2566 strain
    Strategy for constructing a self-excisable RhCMV BAC. (A) Cloning of the full-length RhCMV genome into a BAC vector by Cre/lox recombination. The RhCMV genome structure with expansion of the US1-to-US3 region of recombinant viruses is diagrammed. The open box represents the internal junction between L and S components of the RhCMV genome. Cre/lox recombination was performed in Telo-RF cells. Recombinant clones containing the BAC vector in the viral genome (vRhCMV/BAC-EGFP) were plaque purified, and circular-form viral DNA was transformed into E. coli strain <t>DH10B</t> for plasmid isolation. (B) Substitution of the cre ORF for the egfp ORF by allelic exchange in E. coli . Only a portion of each plasmid is illustrated. The homologous flanking regions and the cre ORF of the recombination vector, pWC212, are illustrated. UL, unique sequences of the L component; US, unique sequences of the S component; PSV40, SV40 promoter; poly A, SV40 polyadenylation signal.
    Escherichia Coli Er2566 Strain, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 79/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher escherichia coli strains hb101
    Strategy for constructing a self-excisable RhCMV BAC. (A) Cloning of the full-length RhCMV genome into a BAC vector by Cre/lox recombination. The RhCMV genome structure with expansion of the US1-to-US3 region of recombinant viruses is diagrammed. The open box represents the internal junction between L and S components of the RhCMV genome. Cre/lox recombination was performed in Telo-RF cells. Recombinant clones containing the BAC vector in the viral genome (vRhCMV/BAC-EGFP) were plaque purified, and circular-form viral DNA was transformed into E. coli strain <t>DH10B</t> for plasmid isolation. (B) Substitution of the cre ORF for the egfp ORF by allelic exchange in E. coli . Only a portion of each plasmid is illustrated. The homologous flanking regions and the cre ORF of the recombination vector, pWC212, are illustrated. UL, unique sequences of the L component; US, unique sequences of the S component; PSV40, SV40 promoter; poly A, SV40 polyadenylation signal.
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    Thermo Fisher bioparticles
    Characterization of Macrophages Generated from iPSCs (A) Flow cytometric analysis of human CD45, CD86 on macrophages derived from peripheral monocytes, control iPSCs, and iPSCs transduced with shPromA or shPromM2 (MDM, WT, PromA, and M2, respectively). (B) Flow cytometric analysis of human CD11b, CD11c, HLA-DR, and CCR5 on macrophages derived from iPSCs transduced with shPromA or shPromM2, and control iPSCs (PromA, M2, and WT, respectively). (C) Macrophages (green) derived from iPSCs transduced with shPromA (upper) and shPromA-M2 (lower) were incubated with Alexa Flour 594 Escherichia coli (red). The cells were microscopically observed after incubation for 1 hr. <t>BioParticles</t> were co-localized in macrophages (yellow in merged pictures). Scale bar, 50 μm. (D) Global gene expression of macrophages derived from iPSCs or MDM was analyzed. Heatmaps show the correlation coefficients between samples.
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    Thermo Fisher one shot top10f escherichia coli
    Characterization of Macrophages Generated from iPSCs (A) Flow cytometric analysis of human CD45, CD86 on macrophages derived from peripheral monocytes, control iPSCs, and iPSCs transduced with shPromA or shPromM2 (MDM, WT, PromA, and M2, respectively). (B) Flow cytometric analysis of human CD11b, CD11c, HLA-DR, and CCR5 on macrophages derived from iPSCs transduced with shPromA or shPromM2, and control iPSCs (PromA, M2, and WT, respectively). (C) Macrophages (green) derived from iPSCs transduced with shPromA (upper) and shPromA-M2 (lower) were incubated with Alexa Flour 594 Escherichia coli (red). The cells were microscopically observed after incubation for 1 hr. <t>BioParticles</t> were co-localized in macrophages (yellow in merged pictures). Scale bar, 50 μm. (D) Global gene expression of macrophages derived from iPSCs or MDM was analyzed. Heatmaps show the correlation coefficients between samples.
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    ΦTE-F expresses ToxI RNA during infection. (A) Upper panel; an S1-nuclease protection assay was used to detect ToxI levels from a ToxIN plasmid during ΦTE wt infection, using an antisense probe against the full 5.5 repeat ToxI sequence [46] . The antisense ToxI-probe was first hybridised to 10 µg of total RNA prepared from Pba ToxIN (pMJ4) at different times after ΦTE infection, and then followed by S1-nuclease treatment. Numbers (+) indicate the time (min) after infection or (−) without the addition of phage. Pba ToxIN-FS (pTA47) and Pba serve as positive and negative controls, respectively. A non-hybridized S1-digested probe (+S1) serves as a further negative control. DH5α 1.5 repeats (pTA96), a non-S1 digested probe (−S1) and an in vitro transcribed Hammerhead ribozyme (HHRz), which cleaves itself during transcription, serve as size markers. HHRz was prepared as described previously [45] . Lower panel; Western blot targeting C-terminal FLAG tagged ToxN contained within total protein harvested from Pba ToxIN (pMJ4) at different time points, with (+, left) and without (−, right) phage infection. Time 0 indicates a sample taken immediately after infection. Total protein from Pba ToxIN (pMJ4) (−) serves as positive control. (B) Infection with escape phage ΦTE-F. Levels of ToxI were determined by S1-assay (upper) as described in (A) with and without infection. ToxN levels were estimated by Western blotting (lower) as described in (A). (C) Expression of the ΦTE-F ToxI locus. An S1-nuclease assay targeting ToxI was performed on total RNA of Pba (pBR322) at different times during ΦTE-F infection. Pba ToxIN (pMJ4) and DH5α serve as positive and negative controls, respectively.

    Journal: PLoS Genetics

    Article Title: Viral Evasion of a Bacterial Suicide System by RNA-Based Molecular Mimicry Enables Infectious Altruism

    doi: 10.1371/journal.pgen.1003023

    Figure Lengend Snippet: ΦTE-F expresses ToxI RNA during infection. (A) Upper panel; an S1-nuclease protection assay was used to detect ToxI levels from a ToxIN plasmid during ΦTE wt infection, using an antisense probe against the full 5.5 repeat ToxI sequence [46] . The antisense ToxI-probe was first hybridised to 10 µg of total RNA prepared from Pba ToxIN (pMJ4) at different times after ΦTE infection, and then followed by S1-nuclease treatment. Numbers (+) indicate the time (min) after infection or (−) without the addition of phage. Pba ToxIN-FS (pTA47) and Pba serve as positive and negative controls, respectively. A non-hybridized S1-digested probe (+S1) serves as a further negative control. DH5α 1.5 repeats (pTA96), a non-S1 digested probe (−S1) and an in vitro transcribed Hammerhead ribozyme (HHRz), which cleaves itself during transcription, serve as size markers. HHRz was prepared as described previously [45] . Lower panel; Western blot targeting C-terminal FLAG tagged ToxN contained within total protein harvested from Pba ToxIN (pMJ4) at different time points, with (+, left) and without (−, right) phage infection. Time 0 indicates a sample taken immediately after infection. Total protein from Pba ToxIN (pMJ4) (−) serves as positive control. (B) Infection with escape phage ΦTE-F. Levels of ToxI were determined by S1-assay (upper) as described in (A) with and without infection. ToxN levels were estimated by Western blotting (lower) as described in (A). (C) Expression of the ΦTE-F ToxI locus. An S1-nuclease assay targeting ToxI was performed on total RNA of Pba (pBR322) at different times during ΦTE-F infection. Pba ToxIN (pMJ4) and DH5α serve as positive and negative controls, respectively.

    Article Snippet: All experiments were performed with E. coli strain DH5α (Gibco/BRL) or Pectobacterium atrosepticum SCRI1043 and derivatives thereof.

    Techniques: Infection, Plasmid Preparation, Sequencing, Negative Control, In Vitro, Western Blot, Positive Control, Expressing, Nuclease Assay

    Analysis of pseudo-ToxI as a potential antitoxin. (A) Alignment of the pseudo-ToxI and ToxI RNA sequences. Pseudo-ToxI nucleotides are coloured to match (B) and (C), with the green and purple bases denoting the 5′ and 3′ ends of a single pseudoknot, respectively. Mutated nucleotides in pseudo-ToxI are coloured orange and numbered according to their grouping, whilst the asterisk indicates the additional 3′ nucleotide. The dotted line connecting the U in group 3 indicates the uracil that is deleted in the case of expanded repeats with 2T sequences rather than 3T. (B) Schematic of the ToxI pseudoknot. Each position containing a mutation in the pseudo-ToxI RNA has been bracketed, with the ToxI base separated from the pseudo-ToxI base by a ‘/’. The mutations have been grouped 1–5, according to position, and highlighted in orange, with the 5′ and 3′ termini in green and violet, respectively. Indels, such as U17 that is deleted in some pseudo-ToxI repeats, and the additional A* inserted in all, have been bordered by a dashed line. Base interactions are indicated by black lines, and duplex and triplex base-interactions are bordered in grey. (C) Detail of the ToxIN trimer with each pseudoknot shown either in blue, purple or beige. Each ToxN monomer is shown as a grey surface. The blue pseudoknot is oriented relative to (B). The positions of mutation groups are shown, with the group number encircled in the same colour as the corresponding pseudoknot. The additional nucleotide of group 5 is not visible as this was not in the original solved ToxIN structure. PDB: 2XDB. (D) Pseudo-ToxI cannot protect from ToxN in an over-expression assay. Protection assays were conducted as per Materials and Methods using strains of E. coli DH5α carrying both pTA49 (inducible ToxN) and a second inducible antitoxin vector as shown, including use of pTA100 as a vector-only control, “vector”. Error bars indicate the standard deviation of triplicate data. (E) Protection assays using mutants of ToxI carried out as in (D) with the antitoxin mutations in each construct numbered as per (B). (F) Protection assays carried out as in (D), testing the full escape loci of ΦTE wt, ΦTE-A and ΦTE-F with full ToxI as a positive control. Under these conditions, there was sufficient antitoxin present to inhibit induced ToxN even without specific induction of the ToxI and ΦTE-F constructs.

    Journal: PLoS Genetics

    Article Title: Viral Evasion of a Bacterial Suicide System by RNA-Based Molecular Mimicry Enables Infectious Altruism

    doi: 10.1371/journal.pgen.1003023

    Figure Lengend Snippet: Analysis of pseudo-ToxI as a potential antitoxin. (A) Alignment of the pseudo-ToxI and ToxI RNA sequences. Pseudo-ToxI nucleotides are coloured to match (B) and (C), with the green and purple bases denoting the 5′ and 3′ ends of a single pseudoknot, respectively. Mutated nucleotides in pseudo-ToxI are coloured orange and numbered according to their grouping, whilst the asterisk indicates the additional 3′ nucleotide. The dotted line connecting the U in group 3 indicates the uracil that is deleted in the case of expanded repeats with 2T sequences rather than 3T. (B) Schematic of the ToxI pseudoknot. Each position containing a mutation in the pseudo-ToxI RNA has been bracketed, with the ToxI base separated from the pseudo-ToxI base by a ‘/’. The mutations have been grouped 1–5, according to position, and highlighted in orange, with the 5′ and 3′ termini in green and violet, respectively. Indels, such as U17 that is deleted in some pseudo-ToxI repeats, and the additional A* inserted in all, have been bordered by a dashed line. Base interactions are indicated by black lines, and duplex and triplex base-interactions are bordered in grey. (C) Detail of the ToxIN trimer with each pseudoknot shown either in blue, purple or beige. Each ToxN monomer is shown as a grey surface. The blue pseudoknot is oriented relative to (B). The positions of mutation groups are shown, with the group number encircled in the same colour as the corresponding pseudoknot. The additional nucleotide of group 5 is not visible as this was not in the original solved ToxIN structure. PDB: 2XDB. (D) Pseudo-ToxI cannot protect from ToxN in an over-expression assay. Protection assays were conducted as per Materials and Methods using strains of E. coli DH5α carrying both pTA49 (inducible ToxN) and a second inducible antitoxin vector as shown, including use of pTA100 as a vector-only control, “vector”. Error bars indicate the standard deviation of triplicate data. (E) Protection assays using mutants of ToxI carried out as in (D) with the antitoxin mutations in each construct numbered as per (B). (F) Protection assays carried out as in (D), testing the full escape loci of ΦTE wt, ΦTE-A and ΦTE-F with full ToxI as a positive control. Under these conditions, there was sufficient antitoxin present to inhibit induced ToxN even without specific induction of the ToxI and ΦTE-F constructs.

    Article Snippet: All experiments were performed with E. coli strain DH5α (Gibco/BRL) or Pectobacterium atrosepticum SCRI1043 and derivatives thereof.

    Techniques: Mutagenesis, Over Expression, Plasmid Preparation, Standard Deviation, Construct, Positive Control

    SsaE interacts with the SPI-2 T3SS translocator protein SseB. Interaction of SsaE with SseB was analyzed by pull-down assays using bacterial lysates of Salmonella wild-type strains harboring the plasmid pBAP-FLAG as a negative control (A) or pSsaE-FLAG (B). Samples were taken before the binding of beads (Lysate), after the final washing with beads (Final wash), and after the elution of fraction with FLAG peptide (Elute). Equal amounts of samples were analyzed by SDS-PAGE and Western blotting with anti-FLAG, anti-SseB, and anti-PagC antibodies. (C) GST pull-down assay using GST or GST-SseB and probed for SsaE-FLAG. Bacterial lysate of E. coli strain DH5α harboring pSsaE-FLAG was incubated with GST or GST-SseB immobilized glutathione Sepharose beads. Samples from lysates before the addition of beads (Lysate) and proteins retained on beads (Elute) were analyzed by SDS-PAGE and Western blotting with anti-FLAG and anti-GST antibodies. A silver-stained SDS-PAGE gel of the elution fractions from pull-down experiments shows the proteins in each sample (bottom).

    Journal: Journal of Bacteriology

    Article Title: Functional Characterization of SsaE, a Novel Chaperone Protein of the Type III Secretion System Encoded by Salmonella Pathogenicity Island 2 ▿

    doi: 10.1128/JB.00863-09

    Figure Lengend Snippet: SsaE interacts with the SPI-2 T3SS translocator protein SseB. Interaction of SsaE with SseB was analyzed by pull-down assays using bacterial lysates of Salmonella wild-type strains harboring the plasmid pBAP-FLAG as a negative control (A) or pSsaE-FLAG (B). Samples were taken before the binding of beads (Lysate), after the final washing with beads (Final wash), and after the elution of fraction with FLAG peptide (Elute). Equal amounts of samples were analyzed by SDS-PAGE and Western blotting with anti-FLAG, anti-SseB, and anti-PagC antibodies. (C) GST pull-down assay using GST or GST-SseB and probed for SsaE-FLAG. Bacterial lysate of E. coli strain DH5α harboring pSsaE-FLAG was incubated with GST or GST-SseB immobilized glutathione Sepharose beads. Samples from lysates before the addition of beads (Lysate) and proteins retained on beads (Elute) were analyzed by SDS-PAGE and Western blotting with anti-FLAG and anti-GST antibodies. A silver-stained SDS-PAGE gel of the elution fractions from pull-down experiments shows the proteins in each sample (bottom).

    Article Snippet: E. coli strains DH5α (Gibco BRL) and MC1061 ( ) were used for molecular cloning and expression of recombinant proteins.

    Techniques: Plasmid Preparation, Negative Control, Binding Assay, SDS Page, Western Blot, Pull Down Assay, Incubation, Staining

    MotB is toxic to E. coli. Heterologous expression of motB in ( a ) BL21(DE3) or ( b ) TOP10F’ E. coli causes cell lysis or inhibits growth, respectively. Cell lines containing either the empty vector pNW129 (circles) or expression vector pNW129-MotB (squares) were induced with 0.2% ( w / v ) arabinose (final concentration) at an OD 600 between 0.3 and 0.4. Growth curves are representative of three independent replicates. To monitor the amount of MotB produced, cell aliquots at 0, 60 and 90 min post-induction were analyzed by SDS-PAGE. The protein gel slice containing MotB is shown for a representative experiment with the band corresponding to MotB designated by a black arrow. It should be noted that although MotB production is seen at 60 min for BL21(DE3)/pNW129-MotB, no additional accumulation is apparent at 90 min perhaps because of the significant cell death at this time point.

    Journal: Viruses

    Article Title: The Bacteriophage T4 MotB Protein, a DNA-Binding Protein, Improves Phage Fitness

    doi: 10.3390/v10070343

    Figure Lengend Snippet: MotB is toxic to E. coli. Heterologous expression of motB in ( a ) BL21(DE3) or ( b ) TOP10F’ E. coli causes cell lysis or inhibits growth, respectively. Cell lines containing either the empty vector pNW129 (circles) or expression vector pNW129-MotB (squares) were induced with 0.2% ( w / v ) arabinose (final concentration) at an OD 600 between 0.3 and 0.4. Growth curves are representative of three independent replicates. To monitor the amount of MotB produced, cell aliquots at 0, 60 and 90 min post-induction were analyzed by SDS-PAGE. The protein gel slice containing MotB is shown for a representative experiment with the band corresponding to MotB designated by a black arrow. It should be noted that although MotB production is seen at 60 min for BL21(DE3)/pNW129-MotB, no additional accumulation is apparent at 90 min perhaps because of the significant cell death at this time point.

    Article Snippet: E. coli strains TOP10F’ (Invitrogen, Carlsbad, CA, USA), BL21(DE3), and BL21(DE3)/pLysE [ ] were used for expression studies.

    Techniques: Expressing, Lysis, Plasmid Preparation, Concentration Assay, Produced, SDS Page

    Barrier RFB1 maps to a 78-bp DNA fragment that is specifically recognized by a binding protein. (A) Diagram of the nontranscribed spacer of the S. pombe rRNA gene, with the locations of the three barriers (RFB1-3) and the replication origin ars3001 indicated. Below the map, lines a to f correspond to the deleted fragments inserted in pIRT2 and assayed as shown in panel C. The names of the resultant plasmids and the results obtained are shown to the right. (B) Map of vector pIRT2 indicating the site where the fragments shown in panel A were cloned (insert). (C) RFB1 activity of fragments a to f in wild-type strain 35 (panels a to f) and RFB1 activity of fragment e in swi1 (EN3182) and swi3 (EN3366) mutant strains (panels e- swi1 and e- swi3 ). Arrows point to the signals corresponding to Y-shaped accumulated replication intermediates with the fork arrested at RFB1. (D) EMSA using labeled fragment e and the indicated amounts of protein extract. In lane 6, a 166-fold excess of unlabeled fragment e was added to the binding reaction. (E) Expression of Sap1p in E. coli TOP10 cells was induced by the addition of 0.02% arabinose to the culture for 2 h. Proteins from 2.5 × 10 8 cells were separated in 12% SDS-polyacrylamide gels (lanes 1 and 2) and transferred to polyvinylidene difluoride membranes, and His 6 -Sap1p was detected with anti-His 6 -peroxidase antibody (Roche) (lanes 3 and 4). Lanes 5 and 6 correspond to mobility shift assays using 4.7 μg of a protein extract from noninduced (−) or induced (+) cells and the same fragment as that used for panel D.

    Journal: Molecular and Cellular Biology

    Article Title: The Mating Type Switch-Activating Protein Sap1 Is Required for Replication Fork Arrest at the rRNA Genes of Fission Yeast

    doi: 10.1128/MCB.25.19.8755-8761.2005

    Figure Lengend Snippet: Barrier RFB1 maps to a 78-bp DNA fragment that is specifically recognized by a binding protein. (A) Diagram of the nontranscribed spacer of the S. pombe rRNA gene, with the locations of the three barriers (RFB1-3) and the replication origin ars3001 indicated. Below the map, lines a to f correspond to the deleted fragments inserted in pIRT2 and assayed as shown in panel C. The names of the resultant plasmids and the results obtained are shown to the right. (B) Map of vector pIRT2 indicating the site where the fragments shown in panel A were cloned (insert). (C) RFB1 activity of fragments a to f in wild-type strain 35 (panels a to f) and RFB1 activity of fragment e in swi1 (EN3182) and swi3 (EN3366) mutant strains (panels e- swi1 and e- swi3 ). Arrows point to the signals corresponding to Y-shaped accumulated replication intermediates with the fork arrested at RFB1. (D) EMSA using labeled fragment e and the indicated amounts of protein extract. In lane 6, a 166-fold excess of unlabeled fragment e was added to the binding reaction. (E) Expression of Sap1p in E. coli TOP10 cells was induced by the addition of 0.02% arabinose to the culture for 2 h. Proteins from 2.5 × 10 8 cells were separated in 12% SDS-polyacrylamide gels (lanes 1 and 2) and transferred to polyvinylidene difluoride membranes, and His 6 -Sap1p was detected with anti-His 6 -peroxidase antibody (Roche) (lanes 3 and 4). Lanes 5 and 6 correspond to mobility shift assays using 4.7 μg of a protein extract from noninduced (−) or induced (+) cells and the same fragment as that used for panel D.

    Article Snippet: The plasmid obtained was used to transform the E. coli strain TOP10 (Invitrogen), and the expression of Sap1p was induced by the addition of 0.02% arabinose to exponentially growing cells for 2 h. The total proteins from 2.5 × 108 cells were separated in 12% sodium dodecyl sulfate (SDS)-polyacrylamide gels and transferred to polyvinylidene difluoride membranes, and His6 -Sap1p was detected by using anti-His6 -peroxidase antibody (Roche).

    Techniques: Binding Assay, Plasmid Preparation, Activity Assay, Mutagenesis, Labeling, Expressing, Mobility Shift

    RNase-L mediates IFNβ production by BMDM in response to bacterial RNA. (A) BMDM were infected with Top10 or LF82 strains of E. coli at an MOI of 5 or 20 for 8 hours and IFNβ was measured by ELISA. (B) BMDMs were transfected with E. coli

    Journal: Inflammatory bowel diseases

    Article Title: RNase-L deficiency exacerbates experimental colitis and colitis-associated cancer

    doi: 10.1097/MIB.0b013e318281f2fd

    Figure Lengend Snippet: RNase-L mediates IFNβ production by BMDM in response to bacterial RNA. (A) BMDM were infected with Top10 or LF82 strains of E. coli at an MOI of 5 or 20 for 8 hours and IFNβ was measured by ELISA. (B) BMDMs were transfected with E. coli

    Article Snippet: E. coli strains TOP10 (Life Technologies) or LF82 were grown to exponential phase in L broth and used at multiplicities of infections of 5 and 20 to infect bone marrow macrophages in bone marrow cultured media (in the absence of antibiotics) for 1.5 hr after which gentamycin (Life Technologies) was added to the media at a concentration of 100 μg/ml.

    Techniques: Infection, Enzyme-linked Immunosorbent Assay, Transfection

    Protein analysis of pET-SUMO-E2-T1 expression in E. coli BL21 (DE3) . (A) Protein from soluble and insoluble fractions separated by electrophoresis on 10% Bis-Tris gel and stained with Coomassie blue. Lane 1, SeeBlue ® Plus2 MW standard; lane 2, soluble protein fraction at 0 hours; lane 3, insoluble protein fraction at 0 hours; lane 4, soluble protein fraction 2 hours post IPTG induction; lane 5, insoluble protein fraction 2 hours IPTG post induction; lane 6, E2-T1 purified from IB (0.4 μL); lane 7, E2-T1 purified from IB (2 μL). (B) Western blot image of E2-T1 protein expressed in E. coli BL21 (DE3). Equivalent amounts of protein from the soluble and insoluble fractions were transferred to Hybond C and incubated with an anti-his antibody and detected by ECL. Lane 1, soluble protein fraction 0 hours; lane 2, insoluble protein fraction 0 hours; lane 3, soluble protein fraction 2 hours post IPTG induction; lane 4, insoluble protein fraction 2 hours post IPTG induction.

    Journal: Microbial Cell Factories

    Article Title: Endotoxin-free purification for the isolation of Bovine Viral Diarrhoea Virus E2 protein from insoluble inclusion body aggregates

    doi: 10.1186/1475-2859-10-57

    Figure Lengend Snippet: Protein analysis of pET-SUMO-E2-T1 expression in E. coli BL21 (DE3) . (A) Protein from soluble and insoluble fractions separated by electrophoresis on 10% Bis-Tris gel and stained with Coomassie blue. Lane 1, SeeBlue ® Plus2 MW standard; lane 2, soluble protein fraction at 0 hours; lane 3, insoluble protein fraction at 0 hours; lane 4, soluble protein fraction 2 hours post IPTG induction; lane 5, insoluble protein fraction 2 hours IPTG post induction; lane 6, E2-T1 purified from IB (0.4 μL); lane 7, E2-T1 purified from IB (2 μL). (B) Western blot image of E2-T1 protein expressed in E. coli BL21 (DE3). Equivalent amounts of protein from the soluble and insoluble fractions were transferred to Hybond C and incubated with an anti-his antibody and detected by ECL. Lane 1, soluble protein fraction 0 hours; lane 2, insoluble protein fraction 0 hours; lane 3, soluble protein fraction 2 hours post IPTG induction; lane 4, insoluble protein fraction 2 hours post IPTG induction.

    Article Snippet: Positive clones were confirmed by sequencing (AGRF, Brisbane, Australia) and transformed into E. coli strain BL21 (DE3, Invitrogen) cells for protein expression.

    Techniques: Positron Emission Tomography, Expressing, Electrophoresis, Staining, Purification, Western Blot, Incubation

    Growth curves were plotted from E. coli BL21 cells transformed with pET-His-IscS ( filled triangle ) and pET-His ( filled square ) and grown aerobically in LB medium supplemented with 2.5 % sodium thiosulfate

    Journal: Indian Journal of Microbiology

    Article Title: Proteomic Analysis of Differential Protein Expression in Acidithiobacillus ferrooxidans Grown on Ferrous Iron or Elemental Sulfur

    doi: 10.1007/s12088-012-0322-7

    Figure Lengend Snippet: Growth curves were plotted from E. coli BL21 cells transformed with pET-His-IscS ( filled triangle ) and pET-His ( filled square ) and grown aerobically in LB medium supplemented with 2.5 % sodium thiosulfate

    Article Snippet: The E. coli BL21 strain (Invitrogen) were transformed with either pET-His-IscS or pET-His, the vector control.

    Techniques: Transformation Assay, Positron Emission Tomography

    Expression and purification of recombinant D1ED III. (A) The DNA sequence of the D1ED III gene was derived using codon usage of Escherichia coli and fully synthesized using the assembly PCR method. The PCR product was cloned into the pET22b vector for recombinant D1ED III expression. The recombinant protein contained an additional HHHHHH sequence (HisTag) at the C-terminus and was expressed under the control of the T7 promoter. (B)The recombinant D1ED III protein purification process was monitored using 17.5% reducing SDS-PAGE followed stained by Coomassie Blue staining and immunoblotting using anti-HisTag antibodies. The recombinant D1ED III was expressed in Escherichia coli strain BL21 (DE3). Lane 1, recombinant D1ED III expression after IPTG induction; lane 2, protein expression in the absence of IPTG induction; lane 3, soluble fraction of recombinant D1ED III; lane 4, purified recombinant D1ED III. Lanes 5–8 show immunoblotting to monitor the recombinant D1ED III induction and purification processes, and the samples in these lanes are the same as those in lanes 1–4, respectively. The arrows indicate the electrophoretic positions of recombinant D1ED III in the gels or blots. (C) Recombinant D1ED III was digested with trypsin to assess their peptide mass fingerprinting. The results confirmed that the major peaks in the mass spectra were derived from recombinant D1ED III.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Dengue-1 Envelope Protein Domain III along with PELC and CpG Oligodeoxynucleotides Synergistically Enhances Immune Responses

    doi: 10.1371/journal.pntd.0001645

    Figure Lengend Snippet: Expression and purification of recombinant D1ED III. (A) The DNA sequence of the D1ED III gene was derived using codon usage of Escherichia coli and fully synthesized using the assembly PCR method. The PCR product was cloned into the pET22b vector for recombinant D1ED III expression. The recombinant protein contained an additional HHHHHH sequence (HisTag) at the C-terminus and was expressed under the control of the T7 promoter. (B)The recombinant D1ED III protein purification process was monitored using 17.5% reducing SDS-PAGE followed stained by Coomassie Blue staining and immunoblotting using anti-HisTag antibodies. The recombinant D1ED III was expressed in Escherichia coli strain BL21 (DE3). Lane 1, recombinant D1ED III expression after IPTG induction; lane 2, protein expression in the absence of IPTG induction; lane 3, soluble fraction of recombinant D1ED III; lane 4, purified recombinant D1ED III. Lanes 5–8 show immunoblotting to monitor the recombinant D1ED III induction and purification processes, and the samples in these lanes are the same as those in lanes 1–4, respectively. The arrows indicate the electrophoretic positions of recombinant D1ED III in the gels or blots. (C) Recombinant D1ED III was digested with trypsin to assess their peptide mass fingerprinting. The results confirmed that the major peaks in the mass spectra were derived from recombinant D1ED III.

    Article Snippet: The Escherichia coli strain BL21 (DE3) (Invitrogen, Carlsbad, CA) was transformed with the expression plasmid pDen1E3 for protein expression.

    Techniques: Expressing, Purification, Recombinant, Sequencing, Derivative Assay, Synthesized, Polymerase Cycling Assembly, Polymerase Chain Reaction, Clone Assay, Plasmid Preparation, Protein Purification, SDS Page, Staining, Peptide Mass Fingerprinting

    Strategy for constructing a self-excisable RhCMV BAC. (A) Cloning of the full-length RhCMV genome into a BAC vector by Cre/lox recombination. The RhCMV genome structure with expansion of the US1-to-US3 region of recombinant viruses is diagrammed. The open box represents the internal junction between L and S components of the RhCMV genome. Cre/lox recombination was performed in Telo-RF cells. Recombinant clones containing the BAC vector in the viral genome (vRhCMV/BAC-EGFP) were plaque purified, and circular-form viral DNA was transformed into E. coli strain DH10B for plasmid isolation. (B) Substitution of the cre ORF for the egfp ORF by allelic exchange in E. coli . Only a portion of each plasmid is illustrated. The homologous flanking regions and the cre ORF of the recombination vector, pWC212, are illustrated. UL, unique sequences of the L component; US, unique sequences of the S component; PSV40, SV40 promoter; poly A, SV40 polyadenylation signal.

    Journal: Journal of Virology

    Article Title: Cloning of the Full-Length Rhesus Cytomegalovirus Genome as an Infectious and Self-Excisable Bacterial Artificial Chromosome for Analysis of Viral Pathogenesis

    doi: 10.1128/JVI.77.9.5073-5083.2003

    Figure Lengend Snippet: Strategy for constructing a self-excisable RhCMV BAC. (A) Cloning of the full-length RhCMV genome into a BAC vector by Cre/lox recombination. The RhCMV genome structure with expansion of the US1-to-US3 region of recombinant viruses is diagrammed. The open box represents the internal junction between L and S components of the RhCMV genome. Cre/lox recombination was performed in Telo-RF cells. Recombinant clones containing the BAC vector in the viral genome (vRhCMV/BAC-EGFP) were plaque purified, and circular-form viral DNA was transformed into E. coli strain DH10B for plasmid isolation. (B) Substitution of the cre ORF for the egfp ORF by allelic exchange in E. coli . Only a portion of each plasmid is illustrated. The homologous flanking regions and the cre ORF of the recombination vector, pWC212, are illustrated. UL, unique sequences of the L component; US, unique sequences of the S component; PSV40, SV40 promoter; poly A, SV40 polyadenylation signal.

    Article Snippet: After incubation at 4°C for 24 h, cellular DNA and proteins were precipitated by centrifugation at 15,000 × g and 4°C for 30 min. DNA in the supernatant was extracted three times with phenol-chloroform, ethanol precipitated, and transformed into E. coli strain DH10B (Invitrogen) by electroporation according to published methods ( ).

    Techniques: BAC Assay, Clone Assay, Plasmid Preparation, Recombinant, Purification, Transformation Assay, Isolation

    Characterization of Macrophages Generated from iPSCs (A) Flow cytometric analysis of human CD45, CD86 on macrophages derived from peripheral monocytes, control iPSCs, and iPSCs transduced with shPromA or shPromM2 (MDM, WT, PromA, and M2, respectively). (B) Flow cytometric analysis of human CD11b, CD11c, HLA-DR, and CCR5 on macrophages derived from iPSCs transduced with shPromA or shPromM2, and control iPSCs (PromA, M2, and WT, respectively). (C) Macrophages (green) derived from iPSCs transduced with shPromA (upper) and shPromA-M2 (lower) were incubated with Alexa Flour 594 Escherichia coli (red). The cells were microscopically observed after incubation for 1 hr. BioParticles were co-localized in macrophages (yellow in merged pictures). Scale bar, 50 μm. (D) Global gene expression of macrophages derived from iPSCs or MDM was analyzed. Heatmaps show the correlation coefficients between samples.

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Generation of HIV-Resistant Macrophages from IPSCs by Using Transcriptional Gene Silencing and Promoter-Targeted RNA

    doi: 10.1016/j.omtn.2018.07.017

    Figure Lengend Snippet: Characterization of Macrophages Generated from iPSCs (A) Flow cytometric analysis of human CD45, CD86 on macrophages derived from peripheral monocytes, control iPSCs, and iPSCs transduced with shPromA or shPromM2 (MDM, WT, PromA, and M2, respectively). (B) Flow cytometric analysis of human CD11b, CD11c, HLA-DR, and CCR5 on macrophages derived from iPSCs transduced with shPromA or shPromM2, and control iPSCs (PromA, M2, and WT, respectively). (C) Macrophages (green) derived from iPSCs transduced with shPromA (upper) and shPromA-M2 (lower) were incubated with Alexa Flour 594 Escherichia coli (red). The cells were microscopically observed after incubation for 1 hr. BioParticles were co-localized in macrophages (yellow in merged pictures). Scale bar, 50 μm. (D) Global gene expression of macrophages derived from iPSCs or MDM was analyzed. Heatmaps show the correlation coefficients between samples.

    Article Snippet: Analysis of Phagocytosis Function in Myeloid Lineage Cells Derived from iPSCs Macrophages derived from iPSCs were incubated for 1 hr with BioParticles (E. coli strain K-12 BioParticles, Alexa Flour 594 conjugated, catalog [Cat] #E-23370; Life Technologies), washed three times with PBS, and observed using an IX71 inverted microscope (Olympus).

    Techniques: Generated, Flow Cytometry, Derivative Assay, Transduction, Incubation, Expressing