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Programmed emergence of an RFP-expressing domain across bacterial populations. <t>TOP10</t> E . coli were co-transformed by the RFP reporter plasmid p4g3R, a bicistronic controller plasmid pCRB DRT7VTPLux*(500/250/50), and a sender plasmid pSB1C3 I0500 (LuxI/LasI) encoding either luxI or lasI under control of the arabinose-inducible P BAD/araC promoter. Adjacent populations of the different triple-transformed cell types were incubated on membranes placed on minimal nutrient agar which has or has not been supplemented with 25 mM arabinose. Fluorescence intensity of each quadrant at t = 3000 min (time relative to start of incubation), corrected for background signal present in the absence of arabinose, is plotted against position (genotype boundary between quadrants 12 and 13). Error bars represent the s.d. of average values between the 12 equidistant quadrants on each membrane. Corresponding images captured at t
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1) Product Images from "Programmed hierarchical patterning of bacterial populations"

Article Title: Programmed hierarchical patterning of bacterial populations

Journal: Nature Communications

doi: 10.1038/s41467-018-03069-3

Programmed emergence of an RFP-expressing domain across bacterial populations. TOP10 E . coli were co-transformed by the RFP reporter plasmid p4g3R, a bicistronic controller plasmid pCRB DRT7VTPLux*(500/250/50), and a sender plasmid pSB1C3 I0500 (LuxI/LasI) encoding either luxI or lasI under control of the arabinose-inducible P BAD/araC promoter. Adjacent populations of the different triple-transformed cell types were incubated on membranes placed on minimal nutrient agar which has or has not been supplemented with 25 mM arabinose. Fluorescence intensity of each quadrant at t = 3000 min (time relative to start of incubation), corrected for background signal present in the absence of arabinose, is plotted against position (genotype boundary between quadrants 12 and 13). Error bars represent the s.d. of average values between the 12 equidistant quadrants on each membrane. Corresponding images captured at t
Figure Legend Snippet: Programmed emergence of an RFP-expressing domain across bacterial populations. TOP10 E . coli were co-transformed by the RFP reporter plasmid p4g3R, a bicistronic controller plasmid pCRB DRT7VTPLux*(500/250/50), and a sender plasmid pSB1C3 I0500 (LuxI/LasI) encoding either luxI or lasI under control of the arabinose-inducible P BAD/araC promoter. Adjacent populations of the different triple-transformed cell types were incubated on membranes placed on minimal nutrient agar which has or has not been supplemented with 25 mM arabinose. Fluorescence intensity of each quadrant at t = 3000 min (time relative to start of incubation), corrected for background signal present in the absence of arabinose, is plotted against position (genotype boundary between quadrants 12 and 13). Error bars represent the s.d. of average values between the 12 equidistant quadrants on each membrane. Corresponding images captured at t

Techniques Used: Expressing, Transformation Assay, Plasmid Preparation, Incubation, Fluorescence

Ratiometric characterization of T7RNAP activity. a Comparison of different ratiometric reporter plasmids of T7RNAP activity. Ratiometric reporter plasmids combining different variants of the T7 promoter, 5′-UTRs, and origins of replication (underlined) were introduced into T7 Express E . coli for induction curves) was quantified using a plate fluorometer-based assay (see Methods for details), and is reported relative to the absence of inducer. Error bars represent the standard deviation (s.d.) of average values yielded between three biological replicate experiments performed on different days. b Ratiometric characterization of intact and split variants of T7RNAP. TOP10 E . coli were co-transformed by the highest dynamic range ratiometric reporter p4g3VT tested under a and different controller plasmids encoding intact or fragmented variants of the T7RNAP gene (underlined) under control of the arabinose-inducible P BAD/araC for induction curves), and is reported relative to the absence of inducer. Error bars represent the s.d. of average values yielded between three biological replicate experiments performed on different days
Figure Legend Snippet: Ratiometric characterization of T7RNAP activity. a Comparison of different ratiometric reporter plasmids of T7RNAP activity. Ratiometric reporter plasmids combining different variants of the T7 promoter, 5′-UTRs, and origins of replication (underlined) were introduced into T7 Express E . coli for induction curves) was quantified using a plate fluorometer-based assay (see Methods for details), and is reported relative to the absence of inducer. Error bars represent the standard deviation (s.d.) of average values yielded between three biological replicate experiments performed on different days. b Ratiometric characterization of intact and split variants of T7RNAP. TOP10 E . coli were co-transformed by the highest dynamic range ratiometric reporter p4g3VT tested under a and different controller plasmids encoding intact or fragmented variants of the T7RNAP gene (underlined) under control of the arabinose-inducible P BAD/araC for induction curves), and is reported relative to the absence of inducer. Error bars represent the s.d. of average values yielded between three biological replicate experiments performed on different days

Techniques Used: Activity Assay, Standard Deviation, Transformation Assay

2) Product Images from "Programmed hierarchical patterning of bacterial populations"

Article Title: Programmed hierarchical patterning of bacterial populations

Journal: Nature Communications

doi: 10.1038/s41467-018-03069-3

HSL-responsive surface-based patterning of bacterial gene expression. a Surface-based circuit behavior in response to HSLs present in the growth medium at uniform concentration. TOP10 E . coli co-transformed by the RFP reporter plasmid p4g3R and the improved bicistronic controller plasmid pCRB DRT7VTPLux*250 (see Fig. 3 ) were incubated on membranes printed with hydrophobic ink, placed on minimal agar containing different combinations of 3OC6HSL (25 μM) and 3OC12HSL (1 μM). Images shown were captured at t = 1500 min (time relative to start of incubation). Corresponding corrected fluorescence intensities are shown in Supplementary Figure 9 . b Surface-based AND gate behavior in response to HSL gradients. TOP10 E . coli cells co-transformed by controller and reporter plasmids as above were incubated on membranes placed on minimal agar lacking supplemented HSLs. Instead, aqueous solutions containing 500 μM 3OC6HSL or 200 μM 3OC12HSL, respectively, were spotted next to the cells on either side and left to diffuse into the bacterial population from opposite directions. Images shown were captured at t = 3000 min (time relative to start of incubation). Corrected fluorescence intensities recorded over time are shown in Supplementary Figure 10
Figure Legend Snippet: HSL-responsive surface-based patterning of bacterial gene expression. a Surface-based circuit behavior in response to HSLs present in the growth medium at uniform concentration. TOP10 E . coli co-transformed by the RFP reporter plasmid p4g3R and the improved bicistronic controller plasmid pCRB DRT7VTPLux*250 (see Fig. 3 ) were incubated on membranes printed with hydrophobic ink, placed on minimal agar containing different combinations of 3OC6HSL (25 μM) and 3OC12HSL (1 μM). Images shown were captured at t = 1500 min (time relative to start of incubation). Corresponding corrected fluorescence intensities are shown in Supplementary Figure 9 . b Surface-based AND gate behavior in response to HSL gradients. TOP10 E . coli cells co-transformed by controller and reporter plasmids as above were incubated on membranes placed on minimal agar lacking supplemented HSLs. Instead, aqueous solutions containing 500 μM 3OC6HSL or 200 μM 3OC12HSL, respectively, were spotted next to the cells on either side and left to diffuse into the bacterial population from opposite directions. Images shown were captured at t = 3000 min (time relative to start of incubation). Corrected fluorescence intensities recorded over time are shown in Supplementary Figure 10

Techniques Used: Expressing, Concentration Assay, Transformation Assay, Plasmid Preparation, Incubation, Fluorescence

Programmed emergence of an RFP-expressing domain across bacterial populations. TOP10 E . coli were co-transformed by the RFP reporter plasmid p4g3R, a bicistronic controller plasmid pCRB DRT7VTPLux*(500/250/50), and a sender plasmid pSB1C3 I0500 (LuxI/LasI) encoding either luxI or lasI under control of the arabinose-inducible P BAD/araC promoter. Adjacent populations of the different triple-transformed cell types were incubated on membranes placed on minimal nutrient agar which has or has not been supplemented with 25 mM arabinose. Fluorescence intensity of each quadrant at t = 3000 min (time relative to start of incubation), corrected for background signal present in the absence of arabinose, is plotted against position (genotype boundary between quadrants 12 and 13). Error bars represent the s.d. of average values between the 12 equidistant quadrants on each membrane. Corresponding images captured at t = 3000 min are shown in Supplementary Figure 11
Figure Legend Snippet: Programmed emergence of an RFP-expressing domain across bacterial populations. TOP10 E . coli were co-transformed by the RFP reporter plasmid p4g3R, a bicistronic controller plasmid pCRB DRT7VTPLux*(500/250/50), and a sender plasmid pSB1C3 I0500 (LuxI/LasI) encoding either luxI or lasI under control of the arabinose-inducible P BAD/araC promoter. Adjacent populations of the different triple-transformed cell types were incubated on membranes placed on minimal nutrient agar which has or has not been supplemented with 25 mM arabinose. Fluorescence intensity of each quadrant at t = 3000 min (time relative to start of incubation), corrected for background signal present in the absence of arabinose, is plotted against position (genotype boundary between quadrants 12 and 13). Error bars represent the s.d. of average values between the 12 equidistant quadrants on each membrane. Corresponding images captured at t = 3000 min are shown in Supplementary Figure 11

Techniques Used: Expressing, Transformation Assay, Plasmid Preparation, Incubation, Fluorescence

Ratiometric characterization of T7RNAP activity. a Comparison of different ratiometric reporter plasmids of T7RNAP activity. Ratiometric reporter plasmids combining different variants of the T7 promoter, 5′-UTRs, and origins of replication (underlined) were introduced into T7 Express E . coli . The maximum fold-induction of relative activity from the T7 promoter in response to a range of IPTG concentrations (see Supplementary Figure 1 for induction curves) was quantified using a plate fluorometer-based assay (see Methods for details), and is reported relative to the absence of inducer. Error bars represent the standard deviation (s.d.) of average values yielded between three biological replicate experiments performed on different days. b Ratiometric characterization of intact and split variants of T7RNAP. TOP10 E . coli were co-transformed by the highest dynamic range ratiometric reporter p4g3VT tested under a and different controller plasmids encoding intact or fragmented variants of the T7RNAP gene (underlined) under control of the arabinose-inducible P BAD/araC promoter. The maximum fold-induction of relative activity from the T7 promoter in response to a range of arabinose concentrations was quantified as described above (see Supplementary Figure 2 for induction curves), and is reported relative to the absence of inducer. Error bars represent the s.d. of average values yielded between three biological replicate experiments performed on different days
Figure Legend Snippet: Ratiometric characterization of T7RNAP activity. a Comparison of different ratiometric reporter plasmids of T7RNAP activity. Ratiometric reporter plasmids combining different variants of the T7 promoter, 5′-UTRs, and origins of replication (underlined) were introduced into T7 Express E . coli . The maximum fold-induction of relative activity from the T7 promoter in response to a range of IPTG concentrations (see Supplementary Figure 1 for induction curves) was quantified using a plate fluorometer-based assay (see Methods for details), and is reported relative to the absence of inducer. Error bars represent the standard deviation (s.d.) of average values yielded between three biological replicate experiments performed on different days. b Ratiometric characterization of intact and split variants of T7RNAP. TOP10 E . coli were co-transformed by the highest dynamic range ratiometric reporter p4g3VT tested under a and different controller plasmids encoding intact or fragmented variants of the T7RNAP gene (underlined) under control of the arabinose-inducible P BAD/araC promoter. The maximum fold-induction of relative activity from the T7 promoter in response to a range of arabinose concentrations was quantified as described above (see Supplementary Figure 2 for induction curves), and is reported relative to the absence of inducer. Error bars represent the s.d. of average values yielded between three biological replicate experiments performed on different days

Techniques Used: Activity Assay, Standard Deviation, Transformation Assay

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Clone Assay:

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Centrifugation:

Article Title: Conserved Streptococcus pneumoniae Spirosomes Suggest a Single Type of Transformation Pilus in Competence
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Amplification:

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Synthesized:

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Article Title: Programmed hierarchical patterning of bacterial populations
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Article Title: Ehrlichia chaffeensis TRP47 enters the nucleus via a MYND-binding domain-dependent mechanism and predominantly binds enhancers of host genes associated with signal transduction, cytoskeletal organization, and immune response
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TA Cloning:

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Construct:

Article Title: Conserved Streptococcus pneumoniae Spirosomes Suggest a Single Type of Transformation Pilus in Competence
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Article Title: Ehrlichia chaffeensis TRP47 enters the nucleus via a MYND-binding domain-dependent mechanism and predominantly binds enhancers of host genes associated with signal transduction, cytoskeletal organization, and immune response
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Article Title: Programmed hierarchical patterning of bacterial populations
Article Snippet: All plasmids (listed in Supplementary Table ) were constructed using Gibson assembly ), or synthesized by Integrated DNA Technologies (Coralville, IA, USA), and are available on Addgene. .. All cloning was performed in TOP10 E . coli (Invitrogen, Waltham, MA).

Article Title: Recombinant Forms of Leishmania amazonensis Excreted/Secreted Promastigote Surface Antigen (PSA) Induce Protective Immune Responses in Dogs
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Article Title: Regulation of acetyl-CoA synthetase transcription by the CrbS/R two-component system is conserved in genetically diverse environmental pathogens
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Article Title: Programmed hierarchical patterning of bacterial populations
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Article Title: Novel pathogenic mutations in C1QTNF5 support a dominant negative disease mechanism in late-onset retinal degeneration
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Article Title: Ehrlichia chaffeensis TRP47 enters the nucleus via a MYND-binding domain-dependent mechanism and predominantly binds enhancers of host genes associated with signal transduction, cytoskeletal organization, and immune response
Article Snippet: .. Constructs were transformed into TOP10 E . coli (Invitrogen, Carlsbad, Calif.), and transformants were selected by growth on LB agar with ampicillin. .. PCR products for pAcGFP1-C constructs were cloned into pre-linearized vector using the In-Fusion PCR Cloning Kit (Clontech), and transformants were selected by growth on LB agar with kanamycin.

Article Title: Investigation of interactions between TLR2, MyD88 and TIRAP by bioluminescence resonance energy transfer is hampered by artefacts of protein overexpression
Article Snippet: .. Constructs were purified from TOP10 E . coli (Life Technologies) using an endotoxin-free Maxiprep kit (Qiagen). .. HEK293FT cells (Thermo Fisher Scientific) were maintained in DMEM (Dulbecco’s modified Eagle’s medium, 4.5 g/L D-glucose, 40 mM sodium bicarbonate, 100 U/ml penicillin and 100 μg/ml streptomycin) supplemented with 10% heat-inactivated FCS, at 37°C in a 5% CO2 humidified incubator.

Concentration Assay:

Article Title: Ehrlichia chaffeensis TRP47 enters the nucleus via a MYND-binding domain-dependent mechanism and predominantly binds enhancers of host genes associated with signal transduction, cytoskeletal organization, and immune response
Article Snippet: Expression constructs and site-directed mutagenesis E . chaffeensis genomic DNA was extracted from infected THP-1 cells using the DNeasy Blood & Tissue Kit (QIAGEN, Hilden, Germany), and DNA concentration was determined with a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific). .. Constructs were transformed into TOP10 E . coli (Invitrogen, Carlsbad, Calif.), and transformants were selected by growth on LB agar with ampicillin.

Article Title: Which Way In? The RalF Arf-GEF Orchestrates Rickettsia Host Cell Invasion
Article Snippet: Protein pull-down RalFRt was cloned into the pTrcHisA vector (Life Technologies, see for primer sequences) and transformed into Top10 E . coli cells (Life Technologies). .. E . coli were lysed using Pierce Lysis Buffer in the presence of HALT Protease Inhibitors (Thermo Scientific) and imidazole added to a final concentration of 10 mM.

Article Title: Ehrlichia chaffeensis TRP47 enters the nucleus via a MYND-binding domain-dependent mechanism and predominantly binds enhancers of host genes associated with signal transduction, cytoskeletal organization, and immune response
Article Snippet: E . chaffeensis genomic DNA was extracted from infected THP-1 cells using the DNeasy Blood & Tissue Kit (QIAGEN, Hilden, Germany), and DNA concentration was determined with a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific). .. Constructs were transformed into TOP10 E . coli (Invitrogen, Carlsbad, Calif.), and transformants were selected by growth on LB agar with ampicillin.

Incubation:

Article Title: Conserved Streptococcus pneumoniae Spirosomes Suggest a Single Type of Transformation Pilus in Competence
Article Snippet: .. 90ng of the linearized vector were incubated in 1:1 molar ratio with the adhE-FLAG pcr product in an In-Fusion cloning reaction (Clontech) and transformed into Top10 E . coli cells (Invitrogen). .. Plasmid DNA was purified from individual clones, verified for AdhE-FLAG coding region insertion, and transformed into competence-induced R1501 cells, followed by selection with kanamycin (Kan).

Activity Assay:

Article Title: Programmed hierarchical patterning of bacterial populations
Article Snippet: All cloning was performed in TOP10 E . coli (Invitrogen, Waltham, MA). .. With exception of ratiometric reporter plasmids for T7RNAP activity ( T7 Express E . coli ; New England Biolabs, Ipswich, MA, USA) all analysis was also carried out in TOP10 E . coli .

Article Title: Programmed hierarchical patterning of bacterial populations
Article Snippet: All cloning was performed in TOP10 E . coli (Invitrogen, Waltham, MA). .. With exception of ratiometric reporter plasmids for T7RNAP activity (T7 Express E . coli ; New England Biolabs, Ipswich, MA, USA) all analysis was also carried out in TOP10 E . coli .

Cell Culture:

Article Title: Conserved Streptococcus pneumoniae Spirosomes Suggest a Single Type of Transformation Pilus in Competence
Article Snippet: 90ng of the linearized vector were incubated in 1:1 molar ratio with the adhE-FLAG pcr product in an In-Fusion cloning reaction (Clontech) and transformed into Top10 E . coli cells (Invitrogen). .. The resulting SO007 strain was sequence-verified for the pCEPx-derived adhE-FLAG –KanR cassette recombination [ ] and cultured in CAT medium to OD600 = 0.15.

Expressing:

Article Title: Ehrlichia chaffeensis TRP47 enters the nucleus via a MYND-binding domain-dependent mechanism and predominantly binds enhancers of host genes associated with signal transduction, cytoskeletal organization, and immune response
Article Snippet: Paragraph title: Expression constructs and site-directed mutagenesis ... Constructs were transformed into TOP10 E . coli (Invitrogen, Carlsbad, Calif.), and transformants were selected by growth on LB agar with ampicillin.

Article Title: Which Way In? The RalF Arf-GEF Orchestrates Rickettsia Host Cell Invasion
Article Snippet: Protein pull-down RalFRt was cloned into the pTrcHisA vector (Life Technologies, see for primer sequences) and transformed into Top10 E . coli cells (Life Technologies). .. Protein expression was induced with 1 mM IPTG overnight at 30°C.

Article Title: Recombinant Forms of Leishmania amazonensis Excreted/Secreted Promastigote Surface Antigen (PSA) Induce Protective Immune Responses in Dogs
Article Snippet: The transformed TOP10 E . coli cells (Invitrogen) were screened for the presence of recombinant plasmid with the LaPSA-38S insert by gene-specific PCR and analyzed with Ncol and Notl restriction enzymes. .. The insert was removed by Ncol and Notl digestion and subcloned into the Ncol and Notl insertion site of Leishmania expression vector pF4X1.4sat1 allowing selection with the antibiotic Nourseothricin to create the recombinant pF4X1.4-La PSA-38S plasmid.

Article Title: Vaccination with the Surface Proteins MUL_2232 and MUL_3720 of Mycobacterium ulcerans Induces Antibodies but Fails to Provide Protection against Buruli Ulcer
Article Snippet: .. After propagation of the generated plasmids in Top10 E . coli (Invitrogen), control restriction and sequencing of the plasmids ensured selection of appropriate clones for expression of the proteins. .. Protein expression was induced in E . coli BL21(DE3) strains (Invitrogen) by addition of 1 mM isopropyl thiogalacoside (Calbiochem) for 4 h at 37°C in lysogeny broth (LB) medium supplemented with Ampicillin.

Modification:

Article Title: Vaccination with the Surface Proteins MUL_2232 and MUL_3720 of Mycobacterium ulcerans Induces Antibodies but Fails to Provide Protection against Buruli Ulcer
Article Snippet: Expression of the antigens as recombinant proteins in E . coli was achieved with the pET28a expression system (Novagen, modified to contain an ampicillin selection cassette). .. After propagation of the generated plasmids in Top10 E . coli (Invitrogen), control restriction and sequencing of the plasmids ensured selection of appropriate clones for expression of the proteins.

Transformation Assay:

Article Title: Conserved Streptococcus pneumoniae Spirosomes Suggest a Single Type of Transformation Pilus in Competence
Article Snippet: .. 90ng of the linearized vector were incubated in 1:1 molar ratio with the adhE-FLAG pcr product in an In-Fusion cloning reaction (Clontech) and transformed into Top10 E . coli cells (Invitrogen). .. Plasmid DNA was purified from individual clones, verified for AdhE-FLAG coding region insertion, and transformed into competence-induced R1501 cells, followed by selection with kanamycin (Kan).

Article Title: Ehrlichia chaffeensis TRP47 enters the nucleus via a MYND-binding domain-dependent mechanism and predominantly binds enhancers of host genes associated with signal transduction, cytoskeletal organization, and immune response
Article Snippet: .. Constructs were transformed into TOP10 E . coli (Invitrogen, Carlsbad, Calif.), and transformants were selected by growth on LB agar with ampicillin. .. PCR products for pAcGFP1-C constructs were cloned into pre-linearized vector using the In-Fusion PCR Cloning Kit (Clontech), and transformants were selected by growth on LB agar with kanamycin.

Article Title: Which Way In? The RalF Arf-GEF Orchestrates Rickettsia Host Cell Invasion
Article Snippet: .. Protein pull-down RalFRt was cloned into the pTrcHisA vector (Life Technologies, see for primer sequences) and transformed into Top10 E . coli cells (Life Technologies). .. Protein expression was induced with 1 mM IPTG overnight at 30°C.

Article Title: Recombinant Forms of Leishmania amazonensis Excreted/Secreted Promastigote Surface Antigen (PSA) Induce Protective Immune Responses in Dogs
Article Snippet: .. The transformed TOP10 E . coli cells (Invitrogen) were screened for the presence of recombinant plasmid with the LaPSA-38S insert by gene-specific PCR and analyzed with Ncol and Notl restriction enzymes. .. The insert was removed by Ncol and Notl digestion and subcloned into the Ncol and Notl insertion site of Leishmania expression vector pF4X1.4sat1 allowing selection with the antibiotic Nourseothricin to create the recombinant pF4X1.4-La PSA-38S plasmid.

Article Title: Regulation of acetyl-CoA synthetase transcription by the CrbS/R two-component system is conserved in genetically diverse environmental pathogens
Article Snippet: .. The PCR was performed using the High Fidelity PCR SuperMix (Invitrogen), and the resulting product was gel-purified, TA-cloned into pCR2.1-TOPO, and transformed into TOP10 E . coli cells (Invitrogen). .. Plasmids carrying inserts of the correct size were verified by sequencing (Genewiz, Cambridge, MA).

Article Title: Application of Brown Planthopper Salivary Gland Extract to Rice Plants Induces Systemic Host mRNA Patterns Associated with Nutrient Remobilization
Article Snippet: .. Cloning, transformation and plasmid purification The secondary PCR products of the SSH were cloned into a pCR® 2.1-TOPO vector and transformed into TOP10 E .coli competent cells (Invitrogen) following the manufacturer's instructions. ..

Article Title: Plant X-tender: An extension of the AssemblX system for the assembly and expression of multigene constructs in plants
Article Snippet: .. Appropriate volumes of the SLiCE, NEBuilder HiFi or Gibson DNA assembled products were transformed into TOP10 E . coli (Invitrogen) or NEB5α (NEB) by electroporation or heat shock according to the manufacturer’s protocols. .. Transformed cells were spread on plates containing appropriate antibiotic, ampicillin (100 μg/ml), spectinomycin (75 μg/ml), kanamycin (50 μg/ml) or rifampicin (20 μg/ml).

Article Title: Ehrlichia chaffeensis TRP47 enters the nucleus via a MYND-binding domain-dependent mechanism and predominantly binds enhancers of host genes associated with signal transduction, cytoskeletal organization, and immune response
Article Snippet: .. Constructs were transformed into TOP10 E . coli (Invitrogen, Carlsbad, Calif.), and transformants were selected by growth on LB agar with ampicillin. .. PCR products for pAcGFP1-C constructs were cloned into pre-linearized vector using the In-Fusion PCR Cloning Kit (Clontech), and transformants were selected by growth on LB agar with kanamycin.

Article Title: Plant X-tender: An extension of the AssemblX system for the assembly and expression of multigene constructs in plants
Article Snippet: .. Appropriate volumes of the SLiCE, NEBuilder HiFi or Gibson DNA assembled products were transformed into TOP10 E . coli (Invitrogen) or NEB5α (NEB) by electroporation or heat shock according to the manufacturer’s protocols. .. Transformed cells were spread on plates containing appropriate antibiotic, ampicillin (100 μg/ml), spectinomycin (75 μg/ml), kanamycin (50 μg/ml) or rifampicin (20 μg/ml).

Electroporation:

Article Title: Plant X-tender: An extension of the AssemblX system for the assembly and expression of multigene constructs in plants
Article Snippet: .. Appropriate volumes of the SLiCE, NEBuilder HiFi or Gibson DNA assembled products were transformed into TOP10 E . coli (Invitrogen) or NEB5α (NEB) by electroporation or heat shock according to the manufacturer’s protocols. .. Transformed cells were spread on plates containing appropriate antibiotic, ampicillin (100 μg/ml), spectinomycin (75 μg/ml), kanamycin (50 μg/ml) or rifampicin (20 μg/ml).

Article Title: Plant X-tender: An extension of the AssemblX system for the assembly and expression of multigene constructs in plants
Article Snippet: .. Appropriate volumes of the SLiCE, NEBuilder HiFi or Gibson DNA assembled products were transformed into TOP10 E . coli (Invitrogen) or NEB5α (NEB) by electroporation or heat shock according to the manufacturer’s protocols. .. Transformed cells were spread on plates containing appropriate antibiotic, ampicillin (100 μg/ml), spectinomycin (75 μg/ml), kanamycin (50 μg/ml) or rifampicin (20 μg/ml).

Ligation:

Article Title: Regulation of acetyl-CoA synthetase transcription by the CrbS/R two-component system is conserved in genetically diverse environmental pathogens
Article Snippet: The PCR was performed using the High Fidelity PCR SuperMix (Invitrogen), and the resulting product was gel-purified, TA-cloned into pCR2.1-TOPO, and transformed into TOP10 E . coli cells (Invitrogen). .. The ligation reactions were transformed into E . coli DH5-αλpir for verification.

Infection:

Article Title: Ehrlichia chaffeensis TRP47 enters the nucleus via a MYND-binding domain-dependent mechanism and predominantly binds enhancers of host genes associated with signal transduction, cytoskeletal organization, and immune response
Article Snippet: Expression constructs and site-directed mutagenesis E . chaffeensis genomic DNA was extracted from infected THP-1 cells using the DNeasy Blood & Tissue Kit (QIAGEN, Hilden, Germany), and DNA concentration was determined with a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific). .. Constructs were transformed into TOP10 E . coli (Invitrogen, Carlsbad, Calif.), and transformants were selected by growth on LB agar with ampicillin.

Article Title: Ehrlichia chaffeensis TRP47 enters the nucleus via a MYND-binding domain-dependent mechanism and predominantly binds enhancers of host genes associated with signal transduction, cytoskeletal organization, and immune response
Article Snippet: E . chaffeensis genomic DNA was extracted from infected THP-1 cells using the DNeasy Blood & Tissue Kit (QIAGEN, Hilden, Germany), and DNA concentration was determined with a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific). .. Constructs were transformed into TOP10 E . coli (Invitrogen, Carlsbad, Calif.), and transformants were selected by growth on LB agar with ampicillin.

Generated:

Article Title: Ehrlichia chaffeensis TRP47 enters the nucleus via a MYND-binding domain-dependent mechanism and predominantly binds enhancers of host genes associated with signal transduction, cytoskeletal organization, and immune response
Article Snippet: PCR products were generated from E . chaffeensis genomic DNA with HotMasterMix (5 PRIME, Gaithersburg, Md.) using the following thermal cycling program: 94°C for 2 min; 30 cycles of 94°C for 30 s, annealing temperature (5°C less than the lowest primer Tm) for 30 s, and 65°C for the appropriate extension time (30 s per 500 product base pairs); and 65°C for 7 min. .. Constructs were transformed into TOP10 E . coli (Invitrogen, Carlsbad, Calif.), and transformants were selected by growth on LB agar with ampicillin.

Article Title: Ehrlichia chaffeensis TRP47 enters the nucleus via a MYND-binding domain-dependent mechanism and predominantly binds enhancers of host genes associated with signal transduction, cytoskeletal organization, and immune response
Article Snippet: PCR products were generated from E . chaffeensis genomic DNA with HotMasterMix (5 PRIME, Gaithersburg, Md.) using the following thermal cycling program: 94°C for 2 min; 30 cycles of 94°C for 30 s, annealing temperature (5°C less than the lowest primer Tm) for 30 s, and 65°C for the appropriate extension time (30 s per 500 product base pairs); and 65°C for 7 min. .. Constructs were transformed into TOP10 E . coli (Invitrogen, Carlsbad, Calif.), and transformants were selected by growth on LB agar with ampicillin.

Article Title: Vaccination with the Surface Proteins MUL_2232 and MUL_3720 of Mycobacterium ulcerans Induces Antibodies but Fails to Provide Protection against Buruli Ulcer
Article Snippet: .. After propagation of the generated plasmids in Top10 E . coli (Invitrogen), control restriction and sequencing of the plasmids ensured selection of appropriate clones for expression of the proteins. .. Protein expression was induced in E . coli BL21(DE3) strains (Invitrogen) by addition of 1 mM isopropyl thiogalacoside (Calbiochem) for 4 h at 37°C in lysogeny broth (LB) medium supplemented with Ampicillin.

Overlap Extension Polymerase Chain Reaction:

Article Title: Regulation of acetyl-CoA synthetase transcription by the CrbS/R two-component system is conserved in genetically diverse environmental pathogens
Article Snippet: The AR02 and AR03 primers carry a complementary 18-bp tag that allows for self-annealing during SOE PCR. .. The PCR was performed using the High Fidelity PCR SuperMix (Invitrogen), and the resulting product was gel-purified, TA-cloned into pCR2.1-TOPO, and transformed into TOP10 E . coli cells (Invitrogen).

Plasmid Preparation:

Article Title: Conserved Streptococcus pneumoniae Spirosomes Suggest a Single Type of Transformation Pilus in Competence
Article Snippet: .. 90ng of the linearized vector were incubated in 1:1 molar ratio with the adhE-FLAG pcr product in an In-Fusion cloning reaction (Clontech) and transformed into Top10 E . coli cells (Invitrogen). .. Plasmid DNA was purified from individual clones, verified for AdhE-FLAG coding region insertion, and transformed into competence-induced R1501 cells, followed by selection with kanamycin (Kan).

Article Title: Ehrlichia chaffeensis TRP47 enters the nucleus via a MYND-binding domain-dependent mechanism and predominantly binds enhancers of host genes associated with signal transduction, cytoskeletal organization, and immune response
Article Snippet: PCR products for pGEX-6P-1 constructs were digested with EcoRI and SalI high-fidelity restriction enzymes (New England Biolabs, Ipswich, Mass.) and ligated into digested vector (Fast-Link DNA Ligase; Epicentre, Madison, Wis.). .. Constructs were transformed into TOP10 E . coli (Invitrogen, Carlsbad, Calif.), and transformants were selected by growth on LB agar with ampicillin.

Article Title: Programmed hierarchical patterning of bacterial populations
Article Snippet: Paragraph title: Plasmid construction ... All cloning was performed in TOP10 E . coli (Invitrogen, Waltham, MA).

Article Title: Which Way In? The RalF Arf-GEF Orchestrates Rickettsia Host Cell Invasion
Article Snippet: .. Protein pull-down RalFRt was cloned into the pTrcHisA vector (Life Technologies, see for primer sequences) and transformed into Top10 E . coli cells (Life Technologies). .. Protein expression was induced with 1 mM IPTG overnight at 30°C.

Article Title: Recombinant Forms of Leishmania amazonensis Excreted/Secreted Promastigote Surface Antigen (PSA) Induce Protective Immune Responses in Dogs
Article Snippet: .. The transformed TOP10 E . coli cells (Invitrogen) were screened for the presence of recombinant plasmid with the LaPSA-38S insert by gene-specific PCR and analyzed with Ncol and Notl restriction enzymes. .. The insert was removed by Ncol and Notl digestion and subcloned into the Ncol and Notl insertion site of Leishmania expression vector pF4X1.4sat1 allowing selection with the antibiotic Nourseothricin to create the recombinant pF4X1.4-La PSA-38S plasmid.

Article Title: Application of Brown Planthopper Salivary Gland Extract to Rice Plants Induces Systemic Host mRNA Patterns Associated with Nutrient Remobilization
Article Snippet: .. Cloning, transformation and plasmid purification The secondary PCR products of the SSH were cloned into a pCR® 2.1-TOPO vector and transformed into TOP10 E .coli competent cells (Invitrogen) following the manufacturer's instructions. ..

Article Title: Plant X-tender: An extension of the AssemblX system for the assembly and expression of multigene constructs in plants
Article Snippet: Assembly reaction contained 50–500 ng of linear vector, an appropriate amount of insert DNA in a 1: 2 to 1: 10 vector to insert molar ratio and 2x NEBuilder HiFi or Gibson DNA Assembly Master Mix. .. Appropriate volumes of the SLiCE, NEBuilder HiFi or Gibson DNA assembled products were transformed into TOP10 E . coli (Invitrogen) or NEB5α (NEB) by electroporation or heat shock according to the manufacturer’s protocols.

Article Title: Novel pathogenic mutations in C1QTNF5 support a dominant negative disease mechanism in late-onset retinal degeneration
Article Snippet: .. Recombination reactions were used to transform TOP10 E . coli (Invitrogen), and selection performed using kanamycin (50 μg/mL) and ampicillin (100 μg/mL) for the entry vector and the destination vector respectively. .. Plasmids were isolated using a plasmid miniprep kit (Qiagen), and the cDNA insert was verified by sequencing.

Article Title: Ehrlichia chaffeensis TRP47 enters the nucleus via a MYND-binding domain-dependent mechanism and predominantly binds enhancers of host genes associated with signal transduction, cytoskeletal organization, and immune response
Article Snippet: PCR products for pGEX-6P-1 constructs were digested with EcoRI and SalI high-fidelity restriction enzymes (New England Biolabs, Ipswich, Mass.) and ligated into digested vector (Fast-Link DNA Ligase; Epicentre, Madison, Wis.). .. Constructs were transformed into TOP10 E . coli (Invitrogen, Carlsbad, Calif.), and transformants were selected by growth on LB agar with ampicillin.

Article Title: Plant X-tender: An extension of the AssemblX system for the assembly and expression of multigene constructs in plants
Article Snippet: Assembly reaction contained 50–500 ng of linear vector, an appropriate amount of insert DNA in a 1: 2 to 1: 10 vector to insert molar ratio and 2x NEBuilder HiFi or Gibson DNA Assembly Master Mix. .. Appropriate volumes of the SLiCE, NEBuilder HiFi or Gibson DNA assembled products were transformed into TOP10 E . coli (Invitrogen) or NEB5α (NEB) by electroporation or heat shock according to the manufacturer’s protocols.

Sequencing:

Article Title: Conserved Streptococcus pneumoniae Spirosomes Suggest a Single Type of Transformation Pilus in Competence
Article Snippet: 90ng of the linearized vector were incubated in 1:1 molar ratio with the adhE-FLAG pcr product in an In-Fusion cloning reaction (Clontech) and transformed into Top10 E . coli cells (Invitrogen). .. The resulting SO007 strain was sequence-verified for the pCEPx-derived adhE-FLAG –KanR cassette recombination [ ] and cultured in CAT medium to OD600 = 0.15.

Article Title: Recombinant Forms of Leishmania amazonensis Excreted/Secreted Promastigote Surface Antigen (PSA) Induce Protective Immune Responses in Dogs
Article Snippet: The transformed TOP10 E . coli cells (Invitrogen) were screened for the presence of recombinant plasmid with the LaPSA-38S insert by gene-specific PCR and analyzed with Ncol and Notl restriction enzymes. .. The resulting construct, encoding a full sequence of La PSA-38S secreted protein, was fused to a C-terminus (His6)-tag.

Article Title: Regulation of acetyl-CoA synthetase transcription by the CrbS/R two-component system is conserved in genetically diverse environmental pathogens
Article Snippet: The PCR was performed using the High Fidelity PCR SuperMix (Invitrogen), and the resulting product was gel-purified, TA-cloned into pCR2.1-TOPO, and transformed into TOP10 E . coli cells (Invitrogen). .. Plasmids carrying inserts of the correct size were verified by sequencing (Genewiz, Cambridge, MA).

Article Title: Novel pathogenic mutations in C1QTNF5 support a dominant negative disease mechanism in late-onset retinal degeneration
Article Snippet: Recombination reactions were used to transform TOP10 E . coli (Invitrogen), and selection performed using kanamycin (50 μg/mL) and ampicillin (100 μg/mL) for the entry vector and the destination vector respectively. .. Plasmids were isolated using a plasmid miniprep kit (Qiagen), and the cDNA insert was verified by sequencing.

Article Title: Vaccination with the Surface Proteins MUL_2232 and MUL_3720 of Mycobacterium ulcerans Induces Antibodies but Fails to Provide Protection against Buruli Ulcer
Article Snippet: .. After propagation of the generated plasmids in Top10 E . coli (Invitrogen), control restriction and sequencing of the plasmids ensured selection of appropriate clones for expression of the proteins. .. Protein expression was induced in E . coli BL21(DE3) strains (Invitrogen) by addition of 1 mM isopropyl thiogalacoside (Calbiochem) for 4 h at 37°C in lysogeny broth (LB) medium supplemented with Ampicillin.

Sonication:

Article Title: Which Way In? The RalF Arf-GEF Orchestrates Rickettsia Host Cell Invasion
Article Snippet: Protein pull-down RalFRt was cloned into the pTrcHisA vector (Life Technologies, see for primer sequences) and transformed into Top10 E . coli cells (Life Technologies). .. Lysates were sonicated three times for 20 sec each using setting 6 of a Sonic Dismembranator (Fisher Scientific). mRFP-Arf5 and –Arf6 were expressed in HEK293T cells as described above.

Article Title: Vaccination with the Surface Proteins MUL_2232 and MUL_3720 of Mycobacterium ulcerans Induces Antibodies but Fails to Provide Protection against Buruli Ulcer
Article Snippet: After propagation of the generated plasmids in Top10 E . coli (Invitrogen), control restriction and sequencing of the plasmids ensured selection of appropriate clones for expression of the proteins. .. Protein lysates were produced by dilution of the bacterial pellet in PBS, the addition of lysozyme and sonication.

Recombinant:

Article Title: Recombinant Forms of Leishmania amazonensis Excreted/Secreted Promastigote Surface Antigen (PSA) Induce Protective Immune Responses in Dogs
Article Snippet: .. The transformed TOP10 E . coli cells (Invitrogen) were screened for the presence of recombinant plasmid with the LaPSA-38S insert by gene-specific PCR and analyzed with Ncol and Notl restriction enzymes. .. The insert was removed by Ncol and Notl digestion and subcloned into the Ncol and Notl insertion site of Leishmania expression vector pF4X1.4sat1 allowing selection with the antibiotic Nourseothricin to create the recombinant pF4X1.4-La PSA-38S plasmid.

Article Title: Vaccination with the Surface Proteins MUL_2232 and MUL_3720 of Mycobacterium ulcerans Induces Antibodies but Fails to Provide Protection against Buruli Ulcer
Article Snippet: Paragraph title: Expression and purification of recombinant M . ulcerans proteins ... After propagation of the generated plasmids in Top10 E . coli (Invitrogen), control restriction and sequencing of the plasmids ensured selection of appropriate clones for expression of the proteins.

DNA Extraction:

Article Title: Regulation of acetyl-CoA synthetase transcription by the CrbS/R two-component system is conserved in genetically diverse environmental pathogens
Article Snippet: The upstream and downstream fragments were amplified using the AR01 and AR02 primers and the AR03 and AR04 primers, respectively, from genomic DNA isolated via the Wizard genomic DNA isolation kit (Promega). .. The PCR was performed using the High Fidelity PCR SuperMix (Invitrogen), and the resulting product was gel-purified, TA-cloned into pCR2.1-TOPO, and transformed into TOP10 E . coli cells (Invitrogen).

Nucleic Acid Electrophoresis:

Article Title: Ehrlichia chaffeensis TRP47 enters the nucleus via a MYND-binding domain-dependent mechanism and predominantly binds enhancers of host genes associated with signal transduction, cytoskeletal organization, and immune response
Article Snippet: Correct size was verified using the FlashGel DNA electrophoresis system (Lonza, Basel, Switzerland), and PCR products were purified with the MinElute PCR Purification Kit (Qiagen). .. Constructs were transformed into TOP10 E . coli (Invitrogen, Carlsbad, Calif.), and transformants were selected by growth on LB agar with ampicillin.

Article Title: Ehrlichia chaffeensis TRP47 enters the nucleus via a MYND-binding domain-dependent mechanism and predominantly binds enhancers of host genes associated with signal transduction, cytoskeletal organization, and immune response
Article Snippet: Correct size was verified using the FlashGel DNA electrophoresis system (Lonza, Basel, Switzerland), and PCR products were purified with the MinElute PCR Purification Kit (Qiagen). .. Constructs were transformed into TOP10 E . coli (Invitrogen, Carlsbad, Calif.), and transformants were selected by growth on LB agar with ampicillin.

Mutagenesis:

Article Title: Ehrlichia chaffeensis TRP47 enters the nucleus via a MYND-binding domain-dependent mechanism and predominantly binds enhancers of host genes associated with signal transduction, cytoskeletal organization, and immune response
Article Snippet: Paragraph title: Expression constructs and site-directed mutagenesis ... Constructs were transformed into TOP10 E . coli (Invitrogen, Carlsbad, Calif.), and transformants were selected by growth on LB agar with ampicillin.

Article Title: Novel pathogenic mutations in C1QTNF5 support a dominant negative disease mechanism in late-onset retinal degeneration
Article Snippet: Recombination reactions were used to transform TOP10 E . coli (Invitrogen), and selection performed using kanamycin (50 μg/mL) and ampicillin (100 μg/mL) for the entry vector and the destination vector respectively. .. Mutations were made in the destination vector using the QuikChange Site Directed mutagenesis II kit (Stratagene), according to the manufacturer’s protocol and using the following primers for mutagenesis (p.S163R (c.489C > G) anti-sense 5′-cagatcaaactgcagcctggcccggtagac-3′, sense 5′-gtctaccgggccaggctgcagtttgatctg-3′, p.P188T (c.562C > A), anti-sense 5′-agagcgaggctgtcttgggccaccc-3′, sense 5′-gggtggcccaagacagcctcgctct-3′, p.G216C (c.646 G > T) anti-sense 5′-gctggcatagatgcaaatgtagtcacccacaccc-3′, sense 5′-gggtgtgggtgactacatttgcatctatgccagc-3′, and p.L191P (c.572T > C), anti-sense 5′-ccccccccgagggcgaggctggc-3′, sense 5′-gccagcctcgccctcgggggggg-3′.

Isolation:

Article Title: Regulation of acetyl-CoA synthetase transcription by the CrbS/R two-component system is conserved in genetically diverse environmental pathogens
Article Snippet: The upstream and downstream fragments were amplified using the AR01 and AR02 primers and the AR03 and AR04 primers, respectively, from genomic DNA isolated via the Wizard genomic DNA isolation kit (Promega). .. The PCR was performed using the High Fidelity PCR SuperMix (Invitrogen), and the resulting product was gel-purified, TA-cloned into pCR2.1-TOPO, and transformed into TOP10 E . coli cells (Invitrogen).

Article Title: Novel pathogenic mutations in C1QTNF5 support a dominant negative disease mechanism in late-onset retinal degeneration
Article Snippet: Recombination reactions were used to transform TOP10 E . coli (Invitrogen), and selection performed using kanamycin (50 μg/mL) and ampicillin (100 μg/mL) for the entry vector and the destination vector respectively. .. Plasmids were isolated using a plasmid miniprep kit (Qiagen), and the cDNA insert was verified by sequencing.

Size-exclusion Chromatography:

Article Title: Which Way In? The RalF Arf-GEF Orchestrates Rickettsia Host Cell Invasion
Article Snippet: Protein pull-down RalFRt was cloned into the pTrcHisA vector (Life Technologies, see for primer sequences) and transformed into Top10 E . coli cells (Life Technologies). .. Lysates were sonicated three times for 20 sec each using setting 6 of a Sonic Dismembranator (Fisher Scientific). mRFP-Arf5 and –Arf6 were expressed in HEK293T cells as described above.

Purification:

Article Title: Conserved Streptococcus pneumoniae Spirosomes Suggest a Single Type of Transformation Pilus in Competence
Article Snippet: 90ng of the linearized vector were incubated in 1:1 molar ratio with the adhE-FLAG pcr product in an In-Fusion cloning reaction (Clontech) and transformed into Top10 E . coli cells (Invitrogen). .. Plasmid DNA was purified from individual clones, verified for AdhE-FLAG coding region insertion, and transformed into competence-induced R1501 cells, followed by selection with kanamycin (Kan).

Article Title: Ehrlichia chaffeensis TRP47 enters the nucleus via a MYND-binding domain-dependent mechanism and predominantly binds enhancers of host genes associated with signal transduction, cytoskeletal organization, and immune response
Article Snippet: Correct size was verified using the FlashGel DNA electrophoresis system (Lonza, Basel, Switzerland), and PCR products were purified with the MinElute PCR Purification Kit (Qiagen). .. Constructs were transformed into TOP10 E . coli (Invitrogen, Carlsbad, Calif.), and transformants were selected by growth on LB agar with ampicillin.

Article Title: Recombinant Forms of Leishmania amazonensis Excreted/Secreted Promastigote Surface Antigen (PSA) Induce Protective Immune Responses in Dogs
Article Snippet: The purified PCR product was cloned in pCR2.1-TOPO TA vector using TOPO TA cloning Kit (Invitrogen) according to the manufacturer’s procedures. .. The transformed TOP10 E . coli cells (Invitrogen) were screened for the presence of recombinant plasmid with the LaPSA-38S insert by gene-specific PCR and analyzed with Ncol and Notl restriction enzymes.

Article Title: Investigation of interactions between TLR2, MyD88 and TIRAP by bioluminescence resonance energy transfer is hampered by artefacts of protein overexpression
Article Snippet: .. Constructs were purified from TOP10 E . coli (Life Technologies) using an endotoxin-free Maxiprep kit (Qiagen). .. Cell culture and transfection HEK293FT cells (Thermo Fisher Scientific) were maintained in DMEM (Dulbecco’s modified Eagle’s medium, 4.5 g/L D-glucose, 40 mM sodium bicarbonate, 100 U/ml penicillin and 100 μg/ml streptomycin) supplemented with 10% heat-inactivated FCS, at 37°C in a 5% CO2 humidified incubator.

Article Title: Application of Brown Planthopper Salivary Gland Extract to Rice Plants Induces Systemic Host mRNA Patterns Associated with Nutrient Remobilization
Article Snippet: .. Cloning, transformation and plasmid purification The secondary PCR products of the SSH were cloned into a pCR® 2.1-TOPO vector and transformed into TOP10 E .coli competent cells (Invitrogen) following the manufacturer's instructions. ..

Article Title: Novel pathogenic mutations in C1QTNF5 support a dominant negative disease mechanism in late-onset retinal degeneration
Article Snippet: The C1QTNF5 att B-flanked PCR product was gel purified (Qiagen) and used in a Gateway BP recombination reaction with the donor vector pDONR221, and then shuttled into pDEST/C-SF TAP via Gateway LR gateway recombination as described by the manufacturer (ThermoFisher Scientific). .. Recombination reactions were used to transform TOP10 E . coli (Invitrogen), and selection performed using kanamycin (50 μg/mL) and ampicillin (100 μg/mL) for the entry vector and the destination vector respectively.

Article Title: Ehrlichia chaffeensis TRP47 enters the nucleus via a MYND-binding domain-dependent mechanism and predominantly binds enhancers of host genes associated with signal transduction, cytoskeletal organization, and immune response
Article Snippet: Correct size was verified using the FlashGel DNA electrophoresis system (Lonza, Basel, Switzerland), and PCR products were purified with the MinElute PCR Purification Kit (Qiagen). .. Constructs were transformed into TOP10 E . coli (Invitrogen, Carlsbad, Calif.), and transformants were selected by growth on LB agar with ampicillin.

Article Title: Investigation of interactions between TLR2, MyD88 and TIRAP by bioluminescence resonance energy transfer is hampered by artefacts of protein overexpression
Article Snippet: .. Constructs were purified from TOP10 E . coli (Life Technologies) using an endotoxin-free Maxiprep kit (Qiagen). .. HEK293FT cells (Thermo Fisher Scientific) were maintained in DMEM (Dulbecco’s modified Eagle’s medium, 4.5 g/L D-glucose, 40 mM sodium bicarbonate, 100 U/ml penicillin and 100 μg/ml streptomycin) supplemented with 10% heat-inactivated FCS, at 37°C in a 5% CO2 humidified incubator.

Article Title: Vaccination with the Surface Proteins MUL_2232 and MUL_3720 of Mycobacterium ulcerans Induces Antibodies but Fails to Provide Protection against Buruli Ulcer
Article Snippet: Paragraph title: Expression and purification of recombinant M . ulcerans proteins ... After propagation of the generated plasmids in Top10 E . coli (Invitrogen), control restriction and sequencing of the plasmids ensured selection of appropriate clones for expression of the proteins.

Polymerase Chain Reaction:

Article Title: Conserved Streptococcus pneumoniae Spirosomes Suggest a Single Type of Transformation Pilus in Competence
Article Snippet: .. 90ng of the linearized vector were incubated in 1:1 molar ratio with the adhE-FLAG pcr product in an In-Fusion cloning reaction (Clontech) and transformed into Top10 E . coli cells (Invitrogen). .. Plasmid DNA was purified from individual clones, verified for AdhE-FLAG coding region insertion, and transformed into competence-induced R1501 cells, followed by selection with kanamycin (Kan).

Article Title: Ehrlichia chaffeensis TRP47 enters the nucleus via a MYND-binding domain-dependent mechanism and predominantly binds enhancers of host genes associated with signal transduction, cytoskeletal organization, and immune response
Article Snippet: PCR products for pGEX-6P-1 constructs were digested with EcoRI and SalI high-fidelity restriction enzymes (New England Biolabs, Ipswich, Mass.) and ligated into digested vector (Fast-Link DNA Ligase; Epicentre, Madison, Wis.). .. Constructs were transformed into TOP10 E . coli (Invitrogen, Carlsbad, Calif.), and transformants were selected by growth on LB agar with ampicillin.

Article Title: Recombinant Forms of Leishmania amazonensis Excreted/Secreted Promastigote Surface Antigen (PSA) Induce Protective Immune Responses in Dogs
Article Snippet: .. The transformed TOP10 E . coli cells (Invitrogen) were screened for the presence of recombinant plasmid with the LaPSA-38S insert by gene-specific PCR and analyzed with Ncol and Notl restriction enzymes. .. The insert was removed by Ncol and Notl digestion and subcloned into the Ncol and Notl insertion site of Leishmania expression vector pF4X1.4sat1 allowing selection with the antibiotic Nourseothricin to create the recombinant pF4X1.4-La PSA-38S plasmid.

Article Title: Regulation of acetyl-CoA synthetase transcription by the CrbS/R two-component system is conserved in genetically diverse environmental pathogens
Article Snippet: .. The PCR was performed using the High Fidelity PCR SuperMix (Invitrogen), and the resulting product was gel-purified, TA-cloned into pCR2.1-TOPO, and transformed into TOP10 E . coli cells (Invitrogen). .. Plasmids carrying inserts of the correct size were verified by sequencing (Genewiz, Cambridge, MA).

Article Title: Application of Brown Planthopper Salivary Gland Extract to Rice Plants Induces Systemic Host mRNA Patterns Associated with Nutrient Remobilization
Article Snippet: .. Cloning, transformation and plasmid purification The secondary PCR products of the SSH were cloned into a pCR® 2.1-TOPO vector and transformed into TOP10 E .coli competent cells (Invitrogen) following the manufacturer's instructions. ..

Article Title: Novel pathogenic mutations in C1QTNF5 support a dominant negative disease mechanism in late-onset retinal degeneration
Article Snippet: The C1QTNF5 att B-flanked PCR product was gel purified (Qiagen) and used in a Gateway BP recombination reaction with the donor vector pDONR221, and then shuttled into pDEST/C-SF TAP via Gateway LR gateway recombination as described by the manufacturer (ThermoFisher Scientific). .. Recombination reactions were used to transform TOP10 E . coli (Invitrogen), and selection performed using kanamycin (50 μg/mL) and ampicillin (100 μg/mL) for the entry vector and the destination vector respectively.

Article Title: Ehrlichia chaffeensis TRP47 enters the nucleus via a MYND-binding domain-dependent mechanism and predominantly binds enhancers of host genes associated with signal transduction, cytoskeletal organization, and immune response
Article Snippet: PCR products for pGEX-6P-1 constructs were digested with EcoRI and SalI high-fidelity restriction enzymes (New England Biolabs, Ipswich, Mass.) and ligated into digested vector (Fast-Link DNA Ligase; Epicentre, Madison, Wis.). .. Constructs were transformed into TOP10 E . coli (Invitrogen, Carlsbad, Calif.), and transformants were selected by growth on LB agar with ampicillin.

Article Title: Vaccination with the Surface Proteins MUL_2232 and MUL_3720 of Mycobacterium ulcerans Induces Antibodies but Fails to Provide Protection against Buruli Ulcer
Article Snippet: PCR products were cut by the restriction enzymes NdeI and NotI (New England Biolabs) and subsequently ligated into pET28 to attach an N-terminal 6xHis-tag. .. After propagation of the generated plasmids in Top10 E . coli (Invitrogen), control restriction and sequencing of the plasmids ensured selection of appropriate clones for expression of the proteins.

IA:

Article Title: Programmed hierarchical patterning of bacterial populations
Article Snippet: All plasmids (listed in Supplementary Table ) were constructed using Gibson assembly ), or synthesized by Integrated DNA Technologies (Coralville, IA, USA), and are available on Addgene. .. All cloning was performed in TOP10 E . coli (Invitrogen, Waltham, MA).

Article Title: Programmed hierarchical patterning of bacterial populations
Article Snippet: Plasmid construction All plasmids (listed in Supplementary Table ) were constructed using Gibson assembly with parts obtained from the MIT Registry of Standard Biological Parts ( http://partsregistry.org ), from Addgene ( www.addgene.org ), or synthesized by Integrated DNA Technologies (Coralville, IA, USA), and are available on Addgene. .. All cloning was performed in TOP10 E . coli (Invitrogen, Waltham, MA).

Chloramphenicol Acetyltransferase Assay:

Article Title: Conserved Streptococcus pneumoniae Spirosomes Suggest a Single Type of Transformation Pilus in Competence
Article Snippet: 90ng of the linearized vector were incubated in 1:1 molar ratio with the adhE-FLAG pcr product in an In-Fusion cloning reaction (Clontech) and transformed into Top10 E . coli cells (Invitrogen). .. The resulting SO007 strain was sequence-verified for the pCEPx-derived adhE-FLAG –KanR cassette recombination [ ] and cultured in CAT medium to OD600 = 0.15.

Immu-Puri:

Article Title: Conserved Streptococcus pneumoniae Spirosomes Suggest a Single Type of Transformation Pilus in Competence
Article Snippet: Paragraph title: Immunopurification of spirosomes ... 90ng of the linearized vector were incubated in 1:1 molar ratio with the adhE-FLAG pcr product in an In-Fusion cloning reaction (Clontech) and transformed into Top10 E . coli cells (Invitrogen).

Selection:

Article Title: Conserved Streptococcus pneumoniae Spirosomes Suggest a Single Type of Transformation Pilus in Competence
Article Snippet: 90ng of the linearized vector were incubated in 1:1 molar ratio with the adhE-FLAG pcr product in an In-Fusion cloning reaction (Clontech) and transformed into Top10 E . coli cells (Invitrogen). .. Plasmid DNA was purified from individual clones, verified for AdhE-FLAG coding region insertion, and transformed into competence-induced R1501 cells, followed by selection with kanamycin (Kan).

Article Title: Recombinant Forms of Leishmania amazonensis Excreted/Secreted Promastigote Surface Antigen (PSA) Induce Protective Immune Responses in Dogs
Article Snippet: The transformed TOP10 E . coli cells (Invitrogen) were screened for the presence of recombinant plasmid with the LaPSA-38S insert by gene-specific PCR and analyzed with Ncol and Notl restriction enzymes. .. The insert was removed by Ncol and Notl digestion and subcloned into the Ncol and Notl insertion site of Leishmania expression vector pF4X1.4sat1 allowing selection with the antibiotic Nourseothricin to create the recombinant pF4X1.4-La PSA-38S plasmid.

Article Title: Novel pathogenic mutations in C1QTNF5 support a dominant negative disease mechanism in late-onset retinal degeneration
Article Snippet: .. Recombination reactions were used to transform TOP10 E . coli (Invitrogen), and selection performed using kanamycin (50 μg/mL) and ampicillin (100 μg/mL) for the entry vector and the destination vector respectively. .. Plasmids were isolated using a plasmid miniprep kit (Qiagen), and the cDNA insert was verified by sequencing.

Article Title: Vaccination with the Surface Proteins MUL_2232 and MUL_3720 of Mycobacterium ulcerans Induces Antibodies but Fails to Provide Protection against Buruli Ulcer
Article Snippet: .. After propagation of the generated plasmids in Top10 E . coli (Invitrogen), control restriction and sequencing of the plasmids ensured selection of appropriate clones for expression of the proteins. .. Protein expression was induced in E . coli BL21(DE3) strains (Invitrogen) by addition of 1 mM isopropyl thiogalacoside (Calbiochem) for 4 h at 37°C in lysogeny broth (LB) medium supplemented with Ampicillin.

In Vitro:

Article Title: Plant X-tender: An extension of the AssemblX system for the assembly and expression of multigene constructs in plants
Article Snippet: Paragraph title: In vitro DNA assembly cloning reactions ... Appropriate volumes of the SLiCE, NEBuilder HiFi or Gibson DNA assembled products were transformed into TOP10 E . coli (Invitrogen) or NEB5α (NEB) by electroporation or heat shock according to the manufacturer’s protocols.

Spectrophotometry:

Article Title: Ehrlichia chaffeensis TRP47 enters the nucleus via a MYND-binding domain-dependent mechanism and predominantly binds enhancers of host genes associated with signal transduction, cytoskeletal organization, and immune response
Article Snippet: Expression constructs and site-directed mutagenesis E . chaffeensis genomic DNA was extracted from infected THP-1 cells using the DNeasy Blood & Tissue Kit (QIAGEN, Hilden, Germany), and DNA concentration was determined with a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific). .. Constructs were transformed into TOP10 E . coli (Invitrogen, Carlsbad, Calif.), and transformants were selected by growth on LB agar with ampicillin.

Article Title: Ehrlichia chaffeensis TRP47 enters the nucleus via a MYND-binding domain-dependent mechanism and predominantly binds enhancers of host genes associated with signal transduction, cytoskeletal organization, and immune response
Article Snippet: E . chaffeensis genomic DNA was extracted from infected THP-1 cells using the DNeasy Blood & Tissue Kit (QIAGEN, Hilden, Germany), and DNA concentration was determined with a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific). .. Constructs were transformed into TOP10 E . coli (Invitrogen, Carlsbad, Calif.), and transformants were selected by growth on LB agar with ampicillin.

Produced:

Article Title: Vaccination with the Surface Proteins MUL_2232 and MUL_3720 of Mycobacterium ulcerans Induces Antibodies but Fails to Provide Protection against Buruli Ulcer
Article Snippet: After propagation of the generated plasmids in Top10 E . coli (Invitrogen), control restriction and sequencing of the plasmids ensured selection of appropriate clones for expression of the proteins. .. After screening for high level recombinant protein expression by analysis of small induced cultures, larger amounts of recombinant proteins were produced by selected expression clones.

FLAG-tag:

Article Title: Conserved Streptococcus pneumoniae Spirosomes Suggest a Single Type of Transformation Pilus in Competence
Article Snippet: Briefly, the adhE gene was PCR-amplified using S . pneumoniae R1501 genomic DNA as template and primer pair 5’-GAG GAA GAA ACC ATG TTG AAA GCT ATG GAG GAA AAT ATG GCT GAT AAA AAA AC-3’ and 5’-AAA ATC AAA CGG ATC TTA CTT GTC ATC GTC ATC CTT GTA ATC TTT ACG GCG TCC TGG TCT TTC TTT G-3’, the latter designed to add a C-terminal FLAG tag to the encoded protein. .. 90ng of the linearized vector were incubated in 1:1 molar ratio with the adhE-FLAG pcr product in an In-Fusion cloning reaction (Clontech) and transformed into Top10 E . coli cells (Invitrogen).

DNA Purification:

Article Title: Application of Brown Planthopper Salivary Gland Extract to Rice Plants Induces Systemic Host mRNA Patterns Associated with Nutrient Remobilization
Article Snippet: Cloning, transformation and plasmid purification The secondary PCR products of the SSH were cloned into a pCR® 2.1-TOPO vector and transformed into TOP10 E .coli competent cells (Invitrogen) following the manufacturer's instructions. .. Plasmid DNA purification was carried out using a QIAfilter Plasmid Midi Kit (Qiagen).

Lysis:

Article Title: Conserved Streptococcus pneumoniae Spirosomes Suggest a Single Type of Transformation Pilus in Competence
Article Snippet: 90ng of the linearized vector were incubated in 1:1 molar ratio with the adhE-FLAG pcr product in an In-Fusion cloning reaction (Clontech) and transformed into Top10 E . coli cells (Invitrogen). .. CSP-induced and non-induced cells were pelleted by centrifugation and resuspended in TBS by brief vortexing in the presence of millimeter-sized glass beads for increased cell lysis.

Article Title: Which Way In? The RalF Arf-GEF Orchestrates Rickettsia Host Cell Invasion
Article Snippet: Protein pull-down RalFRt was cloned into the pTrcHisA vector (Life Technologies, see for primer sequences) and transformed into Top10 E . coli cells (Life Technologies). .. E . coli were lysed using Pierce Lysis Buffer in the presence of HALT Protease Inhibitors (Thermo Scientific) and imidazole added to a final concentration of 10 mM.

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  • 93
    Thermo Fisher e coli top10 carrying vector pcr4 topo
    Resistance against sulfonamide antibiotics mediated by SEW2_dhps01, SEW5_dhps01, AEW9_dhps01 , and AEG2_dhps01 . Five microliters of serially diluted E. coli <t>TOP10</t> cultures with starting OD 600 of 0.5 were spotted onto Iso-Sensitest agar plates supplemented with 1000 mg/L sulfamethazine (+ SMZ), 250 mg/L sulfamethoxazole (+ SMX), 250 mg/L sulfadiazine (+ SDZ) or 500 mg/L sulfisoxazole (+ SOX). Iso-Sensitest agar plates with no sulfonamide added (control) were also included. E. coli TOP10 cultures carrying the cloning vector <t>pCR4-TOPO,</t> pCR4_SEW2_dhps01, pCR4_SEW5_dhps01, pCR4_AEW9_dhps01 or pCR4_AEG2_dhps01 were considered.
    E Coli Top10 Carrying Vector Pcr4 Topo, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher top10 escherichia coli
    DNA methylation in the enhancer region regulates perforin transcription. (a) Promoter region sequence containing enhancer in the PRF1 gene was aligned between human and mouse using the VISTA tool. The conservation degree is indicated by the peaks, with coloured peaks (blue for exon and light red for the non‐coding sequence) indicating > 70% conservation. Ten cytosine–phosphate–guanine sites (CpGs) in the enhancer are displayed upstream from the transcription start site (TSS). (b) Sorted CD8 + T cells from healthy donors were cultured initially in the presence of 200 IU/ml recombinant human interleukin (IL)‐2, 5 μg/ml plate‐coated anti‐CD3 and 1 μg/ml anti‐CD28 stimulating antibodies in vitro . 5‐Azacytidine (5‐aza) was added to the culture at a final concentration of 2·5 μM/ml for 48 h, followed by culture medium replacement and incubation for another 48 h. Flow cytometry data demonstrating perforin expression pre‐ and post‐treatment are shown. (c) Perforin – and perforin + CD8 + T cells were sorted. DNA was extracted and bisulfite‐converted from each cell subset, TA‐cloned to pCR4‐TOPO vector and transformed into <t>TOP10</t> <t>Escherichia</t> coli . Colonies were picked from each cell subset and their DNA was sequenced to measure DNA methylation status of the 10 CpGs in the enhancer. The data display rows represent individually sequenced clone. (d) The bar graphs display total DNA methylation percentage in each CpG from clones in (c).
    Top10 Escherichia Coli, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher escherichia coli
    Representative SDS-PAGE, 2-dimensional electrophoresis (2DE), and Western blots showing the workflow for selection of immunoreactive spots. (A) The 1-dimensional SDS-PAGE image of Staphylococcus aureus Newbould 305 trypsinized protein (lane 1) and <t>Escherichia</t> coli trypsinized protein (lane 2). (B) 2DE gel conducted on 7 cm, pH 4 to 7, immobilized pH gradient (IPG) strips of S. aureus Newbould 305 trypsinized protein. Mr = molecular weight. (C) Immunoblot of S. aureus Newbould 305 trypsinized protein using pooled bovine mastitic milk, with immunoreactive regions highlighted in red circles. (D) 2DE gel conducted on 7 cm, pH 4 to 7, IPG strips of Escherichia coli DH10-β trypsinized protein. (E) Immunoblot of E. coli DH10-β trypsinized protein using bovine mastitic milk, with the immunoreactive regions highlighted in red circles. Color version available online.
    Escherichia Coli, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 389 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher e coli top10 cells
    Recombinant expression of rNanI species corresponding to protease-cleaved NanI fragments. rNanI species corresponding to full-length NanI or the major 65-kDa chymotrypsin-cleaved or 60-kDa mouse SI fluid-cleaved NanI fragments were recombinantly expressed by using E. coli <t>Top10</t> cells and the pTrc-HisB expression system. The rNanI species were then affinity enriched from the E. coli culture by using Talon resin and dialyzed overnight against PBS. Panel A shows the dialyzed samples subjected to SDS-PAGE and then either stained with Coomassie blue (left) or Western blotted for NanI (right). The relative amount of each enriched rNanI species was then determined by a protein assay. All experiments were performed in triplicate, and the mean results are shown. Error bars show standard deviations. # indicates a significant ( P
    E Coli Top10 Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


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

    Journal: Frontiers in Microbiology

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

    doi: 10.3389/fmicb.2019.00460

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

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

    Techniques: Clone Assay, Plasmid Preparation

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

    Journal: Frontiers in Microbiology

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

    doi: 10.3389/fmicb.2019.00460

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

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

    Techniques: Derivative Assay, Plasmid Preparation, Clone Assay

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

    Journal: Clinical and Experimental Immunology

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

    doi: 10.1111/cei.13183

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

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

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

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

    Journal: Journal of dairy science

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

    doi: 10.3168/jds.2017-14040

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

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

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

    Recombinant expression of rNanI species corresponding to protease-cleaved NanI fragments. rNanI species corresponding to full-length NanI or the major 65-kDa chymotrypsin-cleaved or 60-kDa mouse SI fluid-cleaved NanI fragments were recombinantly expressed by using E. coli Top10 cells and the pTrc-HisB expression system. The rNanI species were then affinity enriched from the E. coli culture by using Talon resin and dialyzed overnight against PBS. Panel A shows the dialyzed samples subjected to SDS-PAGE and then either stained with Coomassie blue (left) or Western blotted for NanI (right). The relative amount of each enriched rNanI species was then determined by a protein assay. All experiments were performed in triplicate, and the mean results are shown. Error bars show standard deviations. # indicates a significant ( P

    Journal: Infection and Immunity

    Article Title: Native or Proteolytically Activated NanI Sialidase Enhances the Binding and Cytotoxic Activity of Clostridium perfringens Enterotoxin and Beta Toxin

    doi: 10.1128/IAI.00730-17

    Figure Lengend Snippet: Recombinant expression of rNanI species corresponding to protease-cleaved NanI fragments. rNanI species corresponding to full-length NanI or the major 65-kDa chymotrypsin-cleaved or 60-kDa mouse SI fluid-cleaved NanI fragments were recombinantly expressed by using E. coli Top10 cells and the pTrc-HisB expression system. The rNanI species were then affinity enriched from the E. coli culture by using Talon resin and dialyzed overnight against PBS. Panel A shows the dialyzed samples subjected to SDS-PAGE and then either stained with Coomassie blue (left) or Western blotted for NanI (right). The relative amount of each enriched rNanI species was then determined by a protein assay. All experiments were performed in triplicate, and the mean results are shown. Error bars show standard deviations. # indicates a significant ( P

    Article Snippet: E. coli Top10 cells (ThermoFisher) were routinely grown on LB medium (Fisher Scientific) supplemented with 100 μg/ml of ampicillin (Fisher Scientific), as indicated.

    Techniques: Recombinant, Expressing, SDS Page, Staining, Western Blot