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    Thermo Fisher one shot top10
    AQUA Cloning conditions. (a) The DNA fragment encoding the red fluorescent protein mCherry was PCR amplified with flanking extensions of 16, 24, or 32 bp of homologous sequence overlaps to an SV40 promoter-driven mammalian expression vector which was digested, or PCR amplified. AQUA Cloning was performed with pre-incubations on ice, at room temperature (RT), or at 50°C. DNA mixtures were transformed into chemically competent E . coli <t>Top10</t> cells and colonies were obtained the next day. (b) Typical numbers of colony forming units (CFU) obtained from each condition. The highest number of CFU was derived from a pre-incubation at room temperature and with 32 bp of shared homology with PCR originating DNA fragments (arrow). The accuracies for each condition were determined by analytical colony PCR from eight selected clones and were extrapolated to the total number of CFU (grey). Abbreviations: P SV40 , simian virus 40 early promoter.
    One Shot Top10, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 5090 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher e coli top10 competent cells
    Overexpression and purification of the 45-kDa recombinant GDH protein. The protein was overexpressed and purified as described in Materials and Methods. An SDS-10% polyacrylamide gel stained with Coomassie brilliant blue R-250 is shown. Lanes: M, rainbow molecular size marker in kilodaltons; 1, whole-cell lysate of pOT411 transformant of E. coli <t>TOP10</t> uninduced; 2, whole-cell lysate of E. coli transformed with pOT411 and induced with arabinose; 3 and 4, different amounts of the recombinant protein purified from pOT411 transformant of E. coli TOP10.
    E Coli Top10 Competent Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 669 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher one shot top10 escherichia coli
    Overexpression and purification of the 45-kDa recombinant GDH protein. The protein was overexpressed and purified as described in Materials and Methods. An SDS-10% polyacrylamide gel stained with Coomassie brilliant blue R-250 is shown. Lanes: M, rainbow molecular size marker in kilodaltons; 1, whole-cell lysate of pOT411 transformant of E. coli <t>TOP10</t> uninduced; 2, whole-cell lysate of E. coli transformed with pOT411 and induced with arabinose; 3 and 4, different amounts of the recombinant protein purified from pOT411 transformant of E. coli TOP10.
    One Shot Top10 Escherichia Coli, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 172 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher e coli top10
    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 <t>Top10</t> 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.
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    Thermo Fisher growth conditions e coli top10
    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 <t>Top10</t> 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.
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    AQUA Cloning conditions. (a) The DNA fragment encoding the red fluorescent protein mCherry was PCR amplified with flanking extensions of 16, 24, or 32 bp of homologous sequence overlaps to an SV40 promoter-driven mammalian expression vector which was digested, or PCR amplified. AQUA Cloning was performed with pre-incubations on ice, at room temperature (RT), or at 50°C. DNA mixtures were transformed into chemically competent E . coli Top10 cells and colonies were obtained the next day. (b) Typical numbers of colony forming units (CFU) obtained from each condition. The highest number of CFU was derived from a pre-incubation at room temperature and with 32 bp of shared homology with PCR originating DNA fragments (arrow). The accuracies for each condition were determined by analytical colony PCR from eight selected clones and were extrapolated to the total number of CFU (grey). Abbreviations: P SV40 , simian virus 40 early promoter.

    Journal: PLoS ONE

    Article Title: AQUA Cloning: A Versatile and Simple Enzyme-Free Cloning Approach

    doi: 10.1371/journal.pone.0137652

    Figure Lengend Snippet: AQUA Cloning conditions. (a) The DNA fragment encoding the red fluorescent protein mCherry was PCR amplified with flanking extensions of 16, 24, or 32 bp of homologous sequence overlaps to an SV40 promoter-driven mammalian expression vector which was digested, or PCR amplified. AQUA Cloning was performed with pre-incubations on ice, at room temperature (RT), or at 50°C. DNA mixtures were transformed into chemically competent E . coli Top10 cells and colonies were obtained the next day. (b) Typical numbers of colony forming units (CFU) obtained from each condition. The highest number of CFU was derived from a pre-incubation at room temperature and with 32 bp of shared homology with PCR originating DNA fragments (arrow). The accuracies for each condition were determined by analytical colony PCR from eight selected clones and were extrapolated to the total number of CFU (grey). Abbreviations: P SV40 , simian virus 40 early promoter.

    Article Snippet: E . coli strains The following commercially available strains of E . coli were also used for AQUA Cloning: One Shot TOP10 (Invitrogen cat. No. C4040-03, competency: > 109 CFU/μg), NEB5α (NEB, cat. No. C2987I, competency: 1–3 x 109 CFU/μg), NEB10β (NEB, cat. No. C3019I, competency: 1–3 x 109 CFU/μg), BL21 (DE3) (NEB, cat. No. C2527I, competency: 1–5 x 107 CFU/μg), JM109 (Promega, cat. No. L2005, competency: 108 CFU/μg).

    Techniques: Clone Assay, Polymerase Chain Reaction, Amplification, Sequencing, Expressing, Plasmid Preparation, Transformation Assay, Derivative Assay, Incubation

    AQUA Expression—combined cloning and protein expression. (a) Timeline for AQUA Expression. Cloning and production of recombinant protein in E . coli may be performed within 24 h starting with the PCR until the bacteria are harvested the next day. (b) A 3-DNA fragment cloning was performed by inserting the coding sequence for the red fluorescent protein mCherry into a bacterial T7 promoter-driven expression vector. The vector was split into two parts within the resistance gene for the antibiotic spectinomycin. Therefore, only correctly assembled fragments allow cell growth. (c) AQUA Expression in the expression strain BL21 (DE3) results in red colored bacteria due to mCherry protein production, while the TOP10 strain—lacking the required T7 RNA polymerase—remains colorless.

    Journal: PLoS ONE

    Article Title: AQUA Cloning: A Versatile and Simple Enzyme-Free Cloning Approach

    doi: 10.1371/journal.pone.0137652

    Figure Lengend Snippet: AQUA Expression—combined cloning and protein expression. (a) Timeline for AQUA Expression. Cloning and production of recombinant protein in E . coli may be performed within 24 h starting with the PCR until the bacteria are harvested the next day. (b) A 3-DNA fragment cloning was performed by inserting the coding sequence for the red fluorescent protein mCherry into a bacterial T7 promoter-driven expression vector. The vector was split into two parts within the resistance gene for the antibiotic spectinomycin. Therefore, only correctly assembled fragments allow cell growth. (c) AQUA Expression in the expression strain BL21 (DE3) results in red colored bacteria due to mCherry protein production, while the TOP10 strain—lacking the required T7 RNA polymerase—remains colorless.

    Article Snippet: E . coli strains The following commercially available strains of E . coli were also used for AQUA Cloning: One Shot TOP10 (Invitrogen cat. No. C4040-03, competency: > 109 CFU/μg), NEB5α (NEB, cat. No. C2987I, competency: 1–3 x 109 CFU/μg), NEB10β (NEB, cat. No. C3019I, competency: 1–3 x 109 CFU/μg), BL21 (DE3) (NEB, cat. No. C2527I, competency: 1–5 x 107 CFU/μg), JM109 (Promega, cat. No. L2005, competency: 108 CFU/μg).

    Techniques: Expressing, Clone Assay, Recombinant, Polymerase Chain Reaction, Sequencing, Plasmid Preparation

    AQUA Cloning: a dvanced qu ick a ssembly cloning. (a) DNA parts are produced by PCR, or restriction digest (or both). Oligonucleotides are designed to contribute flanking homologous regions to adjacent DNA fragments of optimally 32 bp in length. DNA parts are assembled into a circular plasmid by sequence-determined directionality. (b) AQUA Cloning work-flow. (1) DNA parts are generated by PCR amplification, or derived from an enzymatic digestion. (2) Next, DNA parts are purified by gel-electrophoresis and (3) mixed and simply incubated in water prior to transformation into chemically competent E . coli Top10 cells for in vivo assembly. (4) Finally, obtained colonies are confirmed for correct assembly by standard methods such as analytical PCR, restriction digest, or comprehensive sequencing.

    Journal: PLoS ONE

    Article Title: AQUA Cloning: A Versatile and Simple Enzyme-Free Cloning Approach

    doi: 10.1371/journal.pone.0137652

    Figure Lengend Snippet: AQUA Cloning: a dvanced qu ick a ssembly cloning. (a) DNA parts are produced by PCR, or restriction digest (or both). Oligonucleotides are designed to contribute flanking homologous regions to adjacent DNA fragments of optimally 32 bp in length. DNA parts are assembled into a circular plasmid by sequence-determined directionality. (b) AQUA Cloning work-flow. (1) DNA parts are generated by PCR amplification, or derived from an enzymatic digestion. (2) Next, DNA parts are purified by gel-electrophoresis and (3) mixed and simply incubated in water prior to transformation into chemically competent E . coli Top10 cells for in vivo assembly. (4) Finally, obtained colonies are confirmed for correct assembly by standard methods such as analytical PCR, restriction digest, or comprehensive sequencing.

    Article Snippet: E . coli strains The following commercially available strains of E . coli were also used for AQUA Cloning: One Shot TOP10 (Invitrogen cat. No. C4040-03, competency: > 109 CFU/μg), NEB5α (NEB, cat. No. C2987I, competency: 1–3 x 109 CFU/μg), NEB10β (NEB, cat. No. C3019I, competency: 1–3 x 109 CFU/μg), BL21 (DE3) (NEB, cat. No. C2527I, competency: 1–5 x 107 CFU/μg), JM109 (Promega, cat. No. L2005, competency: 108 CFU/μg).

    Techniques: Clone Assay, Produced, Polymerase Chain Reaction, Plasmid Preparation, Sequencing, Flow Cytometry, Generated, Amplification, Derivative Assay, Purification, Nucleic Acid Electrophoresis, Incubation, Transformation Assay, In Vivo

    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

    (A) Construction of plasmid pCMPG10652. Constitutive promoter P A , gene hpaA , and 4-Hydroxyphenylacetic acid (4-HPA) responsive promoter P BC were copied from plasmid pRA 2 (Prieto and García, 1997 ) and placed in plasmid pFPV25 (Valdivia and Falkow, 1996 ), upstream of gene gfp . This yields GFP production controlled by 4-HPA and 2-Phenylacetic Acid (PAA) concentrations, detected by HpaA. (B) Fluorescence measurements of GFP production of strain pCMPG10652/ E. coli TOP10 in response to a range of 4-HPA concentrations. (C) Fluorescence measurements of GFP production of strain pCMPG10652/ E. coli TOP10 in response to a range of PAA concentrations. Auxin concentrations used in fluorescence measurements: 3 mM, 2 mM, 1.5 mM, 1 mM, 500 μM, 250 μM, 125 μM, 62 μM, 31.25 μM, 15.625 μM, and 0M. n = 8 per measured concentration.

    Journal: Frontiers in Bioengineering and Biotechnology

    Article Title: Design and Construction of a Whole Cell Bacterial 4-Hydroxyphenylacetic Acid and 2-Phenylacetic Acid Bioassay

    doi: 10.3389/fbioe.2015.00088

    Figure Lengend Snippet: (A) Construction of plasmid pCMPG10652. Constitutive promoter P A , gene hpaA , and 4-Hydroxyphenylacetic acid (4-HPA) responsive promoter P BC were copied from plasmid pRA 2 (Prieto and García, 1997 ) and placed in plasmid pFPV25 (Valdivia and Falkow, 1996 ), upstream of gene gfp . This yields GFP production controlled by 4-HPA and 2-Phenylacetic Acid (PAA) concentrations, detected by HpaA. (B) Fluorescence measurements of GFP production of strain pCMPG10652/ E. coli TOP10 in response to a range of 4-HPA concentrations. (C) Fluorescence measurements of GFP production of strain pCMPG10652/ E. coli TOP10 in response to a range of PAA concentrations. Auxin concentrations used in fluorescence measurements: 3 mM, 2 mM, 1.5 mM, 1 mM, 500 μM, 250 μM, 125 μM, 62 μM, 31.25 μM, 15.625 μM, and 0M. n = 8 per measured concentration.

    Article Snippet: All experiments were performed with E. coli TOP10 strains, purchased from Life Technologies Europe.

    Techniques: Plasmid Preparation, Fluorescence, Concentration Assay

    Constructing individual circuits and combinatorial libraries (A) Combinatorial assembly of a 3-part biosynthetic pathway for deoxychromoviridans (an insoluble green alkaloid pigment) by UNS-guided assembly. Each part contained one of six promoters, a triple terminator, and one of the three genes required for deoxychromoviridans biosynthesis ( vioB , vioA , vioE ). (i) Analytical restriction digestion of a pool of 60 clones obtained by this method. The pool was digested to isolate the destination vector backbone (black arrow) from the inserts assembled into it. Indicated are the frequent, correctly-sized (green arrow) and rare, incorrectly-sized (red arrows) inserts. (ii) Transformation of empty pDestET into TOP10 competent cells yields a lawn of unpigmented TOP 10 E. coli . (iii) Transformation of the isothermal assembly reaction (containing the vioBAE library, indicated by “Vio Lib” in the figure) into TOP10 E. coli yields colonies with variable levels of green pigmentation. (B) Construction of individual, four-part circuits for and logical computation in mammalian cells. (i) Parts were assembled into the pDestRmceBAC destination vector, which is capable of single-copy integration into appropriate mammalian cell lines. The first three parts (A, B, C1) were mixed with either the D1 or D2 part. Each mixture was assembled on its own, transformed into E. coli, .

    Journal: Nature protocols

    Article Title: Unique nucleotide sequence (UNS)-guided assembly of repetitive DNA parts for synthetic biology applications

    doi: 10.1038/nprot.2014.145

    Figure Lengend Snippet: Constructing individual circuits and combinatorial libraries (A) Combinatorial assembly of a 3-part biosynthetic pathway for deoxychromoviridans (an insoluble green alkaloid pigment) by UNS-guided assembly. Each part contained one of six promoters, a triple terminator, and one of the three genes required for deoxychromoviridans biosynthesis ( vioB , vioA , vioE ). (i) Analytical restriction digestion of a pool of 60 clones obtained by this method. The pool was digested to isolate the destination vector backbone (black arrow) from the inserts assembled into it. Indicated are the frequent, correctly-sized (green arrow) and rare, incorrectly-sized (red arrows) inserts. (ii) Transformation of empty pDestET into TOP10 competent cells yields a lawn of unpigmented TOP 10 E. coli . (iii) Transformation of the isothermal assembly reaction (containing the vioBAE library, indicated by “Vio Lib” in the figure) into TOP10 E. coli yields colonies with variable levels of green pigmentation. (B) Construction of individual, four-part circuits for and logical computation in mammalian cells. (i) Parts were assembled into the pDestRmceBAC destination vector, which is capable of single-copy integration into appropriate mammalian cell lines. The first three parts (A, B, C1) were mixed with either the D1 or D2 part. Each mixture was assembled on its own, transformed into E. coli, .

    Article Snippet: L-arabinose (Sigma-Aldrich, cat. no. A3256) QIAprep Spin Miniprep Kit (QIAGEN, cat. no. 27104) QIAGEN Plasmid Plus Midi Kit (QIAGEN, cat. no. 12943) One Shot Mach1 or TOP10 chemically competent E. coli (Life Technologies, cat. nos.

    Techniques: Clone Assay, Plasmid Preparation, Transformation Assay

    Overexpression and purification of the 45-kDa recombinant GDH protein. The protein was overexpressed and purified as described in Materials and Methods. An SDS-10% polyacrylamide gel stained with Coomassie brilliant blue R-250 is shown. Lanes: M, rainbow molecular size marker in kilodaltons; 1, whole-cell lysate of pOT411 transformant of E. coli TOP10 uninduced; 2, whole-cell lysate of E. coli transformed with pOT411 and induced with arabinose; 3 and 4, different amounts of the recombinant protein purified from pOT411 transformant of E. coli TOP10.

    Journal: Clinical and Diagnostic Laboratory Immunology

    Article Title: Cloning and Characterization of the Gene Encoding the Glutamate Dehydrogenase of Streptococcus suis Serotype 2

    doi: 10.1128/CDLI.8.2.251-257.2001

    Figure Lengend Snippet: Overexpression and purification of the 45-kDa recombinant GDH protein. The protein was overexpressed and purified as described in Materials and Methods. An SDS-10% polyacrylamide gel stained with Coomassie brilliant blue R-250 is shown. Lanes: M, rainbow molecular size marker in kilodaltons; 1, whole-cell lysate of pOT411 transformant of E. coli TOP10 uninduced; 2, whole-cell lysate of E. coli transformed with pOT411 and induced with arabinose; 3 and 4, different amounts of the recombinant protein purified from pOT411 transformant of E. coli TOP10.

    Article Snippet: The DNA insert in pOT410 was cloned in frame into a pBAD/ Myc -His version B expression vector to create pOT411. pOT411 was transformed into E. coli TOP10-competent cells and overexpressed by following the manufacturer's protocol (Invitrogen).

    Techniques: Over Expression, Purification, Recombinant, Staining, Marker, Transformation Assay

    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.

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gkt253

    Figure Lengend 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.

    Article Snippet: E. coli strain and growth conditions All experiments were conducted with the E. coli Top10 (Invitrogen) strain (F- mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 nupG recA1 araD139 Δ(ara-leu)7697 galE15 galK16 rpsL(StrR ) endA1 λ− ).

    Techniques: 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.

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gkt253

    Figure Lengend 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.

    Article Snippet: E. coli strain and growth conditions All experiments were conducted with the E. coli Top10 (Invitrogen) strain (F- mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 nupG recA1 araD139 Δ(ara-leu)7697 galE15 galK16 rpsL(StrR ) endA1 λ− ).

    Techniques: 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.

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gkt253

    Figure Lengend 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.

    Article Snippet: E. coli strain and growth conditions All experiments were conducted with the E. coli Top10 (Invitrogen) strain (F- mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 nupG recA1 araD139 Δ(ara-leu)7697 galE15 galK16 rpsL(StrR ) endA1 λ− ).

    Techniques: 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.

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gkt253

    Figure Lengend 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.

    Article Snippet: E. coli strain and growth conditions All experiments were conducted with the E. coli Top10 (Invitrogen) strain (F- mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 nupG recA1 araD139 Δ(ara-leu)7697 galE15 galK16 rpsL(StrR ) endA1 λ− ).

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