ppcrscript amp sk(+) vector  (Agilent technologies)

Journal:

Article Title: Identification and Characterization of a Novel Uropathogenic Escherichia coli -Associated Fimbrial Gene Cluster

doi: 10.1128/IAI.72.7.3890-3901.2004

Figure Lengend Snippet: Strains and plasmids used in this study

Article Snippet: Antibiotics were used at the following concentrations unless otherwise indicated: ampicillin, 50 μg/ml; chloramphenicol, 50 μg/ml; kanamycin, 50 μg/ml; nalidixic acid, 50 μg/ml; and rifampin, 50 μg/ml. table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 E. coli strain or plasmid Relevant characteristics Source or reference E. coli strains CFT073 Pyelonephritis isolate, fim + pap + hly + 31 CFT073 Rif Spontaneous rifampin-resistant mutant of CFT073 This study UMD100 CFT073 aufC ::pSSP104, ampicillin resistant This study UMD132 CFT073 Δ( aufABCDEFG ), nalidixic and chloramphenicol resistant This study UMD133 CFT073 Δ( aufABCDEFG ), nalidixic resistant This study ORN172 Δ( fimBEACDFGH ), kanamycin resistant 43 HB101 F − ( gpt-proA ) 62 leu supe44 ara-14 galK2 lacY1 ( mcrC-mrr ) rpsL20 xyl-5 mtl-1 recA13 37 TOP10F′ F′[ lacI q Tn 10 (Tet R )] mrcA Δ( mrr-hsdRMS-mrcrBC ) φ80 lacZ ΔM15 Δφ lacX74 recA1 araD139 Δ( ara-leu )7697 galU galk rpsL (Str r ) endA1 nupG Invitrogen BL21-AI F − ompT hsdS B (r B − m B − ) gal dcm (DE3) araB :: T7RNAP-tet A Invitrogen BL21 (DE3)pLysS F − ompT hsdS B (r B − m B − ) gal dcm (DE3) pLysS (Cam r ) Invitrogen S17-λ pir pro thi recA hsdR; chromosomal RP4-2 (Tn 1 ::ISR 1 tet ::Mu Km::Tn 7 ); λpir 17 Plasmids pcosmid 12A Cosmid clone of CFT073 that contains the entire auf operon pELB1 pCRScript Cam (SK+) with the 8.6-kb BsiWI/KasI fragment containing the auf operon This study pELB2 pBlueScript-II KS(+) with the 9-kb NotI/XhoI fragment containing auf operon under T7 promoter control This study pAufA pCR T7/NT-TOPO with the aufA gene lacking the codons for the signal sequence This study pPCRScript Cam (SK+) Cloning vector, chloramphenicol resistant Stratagene pBlueScript-II KS (+) Cloning vector, ampicillin resistant Stratagene pCR T7/NT-TOPO Cloning vector, ampicillin resistant Invitrogen pGP704 Suicide vector, ampicillin resistant, R6K origin 29 pT-Aadv Cloning vector, ampicillin resistant Clonetech pSSP102 pT-Adv with the 1.2-kb aufC fragment Stratagene pSSP104 pGP704 with the 1.2-kb XabI/KpnI aufC fragment, ampicillin resistant This study PKD46 Low-copy-number plasmid encoding the phage λ Red recombinase expressing γ, β, and exo from the arabinose-inducible Para B 8 pFT-A Helper plasmid, carrying the FLP recombinase, ampicillin resistant 34 pKD3 Plasmid containing an FRT-flanked cat gene, ampicillin and chloramphenicol resistant 8 Open in a separate window Strains and plasmids used in this study Recombinant DNA methods.

Techniques: Plasmid Preparation, Mutagenesis, Sequencing, Clone Assay, Expressing

Journal:

Article Title: Identification and Characterization of a Novel Uropathogenic Escherichia coli -Associated Fimbrial Gene Cluster

doi: 10.1128/IAI.72.7.3890-3901.2004

Figure Lengend Snippet: Overexpression of AufA in E. coli BL21-AI. The auf gene cluster was cloned into pBluescript under the control of the T7 promoter and expressed in BL21-AI cells by using various concentrations of arabinose. Glucose (0.2%) was added to the samples lacking arabinose to repress auf expression. Whole-cell lysates of BL21-AI harboring either pELB2 or the control plasmid pBluescript were separated on a SDS-PAGE and transferred to polyvinylidene difluoride membranes for Western blotting with absorbed AufA antiserum (1:5,000). The prepilin and mature pilin forms of AufA are indicated by arrows. The position and mass of a molecular mass marker are noted on the left side.

Article Snippet: Antibiotics were used at the following concentrations unless otherwise indicated: ampicillin, 50 μg/ml; chloramphenicol, 50 μg/ml; kanamycin, 50 μg/ml; nalidixic acid, 50 μg/ml; and rifampin, 50 μg/ml. table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 E. coli strain or plasmid Relevant characteristics Source or reference E. coli strains CFT073 Pyelonephritis isolate, fim + pap + hly + 31 CFT073 Rif Spontaneous rifampin-resistant mutant of CFT073 This study UMD100 CFT073 aufC ::pSSP104, ampicillin resistant This study UMD132 CFT073 Δ( aufABCDEFG ), nalidixic and chloramphenicol resistant This study UMD133 CFT073 Δ( aufABCDEFG ), nalidixic resistant This study ORN172 Δ( fimBEACDFGH ), kanamycin resistant 43 HB101 F − ( gpt-proA ) 62 leu supe44 ara-14 galK2 lacY1 ( mcrC-mrr ) rpsL20 xyl-5 mtl-1 recA13 37 TOP10F′ F′[ lacI q Tn 10 (Tet R )] mrcA Δ( mrr-hsdRMS-mrcrBC ) φ80 lacZ ΔM15 Δφ lacX74 recA1 araD139 Δ( ara-leu )7697 galU galk rpsL (Str r ) endA1 nupG Invitrogen BL21-AI F − ompT hsdS B (r B − m B − ) gal dcm (DE3) araB :: T7RNAP-tet A Invitrogen BL21 (DE3)pLysS F − ompT hsdS B (r B − m B − ) gal dcm (DE3) pLysS (Cam r ) Invitrogen S17-λ pir pro thi recA hsdR; chromosomal RP4-2 (Tn 1 ::ISR 1 tet ::Mu Km::Tn 7 ); λpir 17 Plasmids pcosmid 12A Cosmid clone of CFT073 that contains the entire auf operon pELB1 pCRScript Cam (SK+) with the 8.6-kb BsiWI/KasI fragment containing the auf operon This study pELB2 pBlueScript-II KS(+) with the 9-kb NotI/XhoI fragment containing auf operon under T7 promoter control This study pAufA pCR T7/NT-TOPO with the aufA gene lacking the codons for the signal sequence This study pPCRScript Cam (SK+) Cloning vector, chloramphenicol resistant Stratagene pBlueScript-II KS (+) Cloning vector, ampicillin resistant Stratagene pCR T7/NT-TOPO Cloning vector, ampicillin resistant Invitrogen pGP704 Suicide vector, ampicillin resistant, R6K origin 29 pT-Aadv Cloning vector, ampicillin resistant Clonetech pSSP102 pT-Adv with the 1.2-kb aufC fragment Stratagene pSSP104 pGP704 with the 1.2-kb XabI/KpnI aufC fragment, ampicillin resistant This study PKD46 Low-copy-number plasmid encoding the phage λ Red recombinase expressing γ, β, and exo from the arabinose-inducible Para B 8 pFT-A Helper plasmid, carrying the FLP recombinase, ampicillin resistant 34 pKD3 Plasmid containing an FRT-flanked cat gene, ampicillin and chloramphenicol resistant 8 Open in a separate window Strains and plasmids used in this study Recombinant DNA methods.

Techniques: Over Expression, Clone Assay, Expressing, Plasmid Preparation, SDS Page, Western Blot, Marker

Journal:

Article Title: Identification and Characterization of a Novel Uropathogenic Escherichia coli -Associated Fimbrial Gene Cluster

doi: 10.1128/IAI.72.7.3890-3901.2004

Figure Lengend Snippet: Surface localization of AufA on E. coli BL21-AI cells. (A) BL21-AI cells harboring either pELB2 or pBluescript were grown under repressed (R) or inducing (I) conditions using 0.2% arabinose. Surface proteins were isolated by heating at 60°C followed by vortexing and centrifugation. Isolated surface proteins (S) and crude cell lysates (P) were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes for Western blotting with absorbed AufA antiserum (1:5,000). (B) Sensitivity of AufA to proteinase K treatment. Induced BL21-AI cells containing the auf operon plasmid were treated with or without proteinase K (200 μg/ml) for 1 h at 37°C followed by the addition of phenylmethylsulfonyl fluoride (1.6 mg/ml). Surface localization was performed as for panel A. Arrows indicate the prepilin and mature pilin forms of AufA.

Article Snippet: Antibiotics were used at the following concentrations unless otherwise indicated: ampicillin, 50 μg/ml; chloramphenicol, 50 μg/ml; kanamycin, 50 μg/ml; nalidixic acid, 50 μg/ml; and rifampin, 50 μg/ml. table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 E. coli strain or plasmid Relevant characteristics Source or reference E. coli strains CFT073 Pyelonephritis isolate, fim + pap + hly + 31 CFT073 Rif Spontaneous rifampin-resistant mutant of CFT073 This study UMD100 CFT073 aufC ::pSSP104, ampicillin resistant This study UMD132 CFT073 Δ( aufABCDEFG ), nalidixic and chloramphenicol resistant This study UMD133 CFT073 Δ( aufABCDEFG ), nalidixic resistant This study ORN172 Δ( fimBEACDFGH ), kanamycin resistant 43 HB101 F − ( gpt-proA ) 62 leu supe44 ara-14 galK2 lacY1 ( mcrC-mrr ) rpsL20 xyl-5 mtl-1 recA13 37 TOP10F′ F′[ lacI q Tn 10 (Tet R )] mrcA Δ( mrr-hsdRMS-mrcrBC ) φ80 lacZ ΔM15 Δφ lacX74 recA1 araD139 Δ( ara-leu )7697 galU galk rpsL (Str r ) endA1 nupG Invitrogen BL21-AI F − ompT hsdS B (r B − m B − ) gal dcm (DE3) araB :: T7RNAP-tet A Invitrogen BL21 (DE3)pLysS F − ompT hsdS B (r B − m B − ) gal dcm (DE3) pLysS (Cam r ) Invitrogen S17-λ pir pro thi recA hsdR; chromosomal RP4-2 (Tn 1 ::ISR 1 tet ::Mu Km::Tn 7 ); λpir 17 Plasmids pcosmid 12A Cosmid clone of CFT073 that contains the entire auf operon pELB1 pCRScript Cam (SK+) with the 8.6-kb BsiWI/KasI fragment containing the auf operon This study pELB2 pBlueScript-II KS(+) with the 9-kb NotI/XhoI fragment containing auf operon under T7 promoter control This study pAufA pCR T7/NT-TOPO with the aufA gene lacking the codons for the signal sequence This study pPCRScript Cam (SK+) Cloning vector, chloramphenicol resistant Stratagene pBlueScript-II KS (+) Cloning vector, ampicillin resistant Stratagene pCR T7/NT-TOPO Cloning vector, ampicillin resistant Invitrogen pGP704 Suicide vector, ampicillin resistant, R6K origin 29 pT-Aadv Cloning vector, ampicillin resistant Clonetech pSSP102 pT-Adv with the 1.2-kb aufC fragment Stratagene pSSP104 pGP704 with the 1.2-kb XabI/KpnI aufC fragment, ampicillin resistant This study PKD46 Low-copy-number plasmid encoding the phage λ Red recombinase expressing γ, β, and exo from the arabinose-inducible Para B 8 pFT-A Helper plasmid, carrying the FLP recombinase, ampicillin resistant 34 pKD3 Plasmid containing an FRT-flanked cat gene, ampicillin and chloramphenicol resistant 8 Open in a separate window Strains and plasmids used in this study Recombinant DNA methods.

Techniques: Isolation, Centrifugation, SDS Page, Western Blot, Plasmid Preparation

Journal:

Article Title: Identification and Characterization of a Novel Uropathogenic Escherichia coli -Associated Fimbrial Gene Cluster

doi: 10.1128/IAI.72.7.3890-3901.2004

Figure Lengend Snippet: Immunoelectron microscopy of BL21-AI cells expressing Auf. BL21-AI cells harboring either the auf gene cluster plasmid pELB2 (A and B) or the control plasmid pBluescript (C and D) were grown under repressing (A and C) or inducing (B and D) conditions with 0.2% arabinose. Bacteria were reacted first with absorbed AufA antiserum (1:20 dilution) and then with anti-rabbit IgG conjugated to 10-nm-diameter gold particles (1:200 dilution) followed by negative staining with phosphotungstic acid. The arrow in panel B indicates the amorphous material extending from the surface of BL21-AI(pELB2)-induced cells bound by gold particles. BL21-AI(pELB2)-repressed cells and BL21-AI(pBluescript)-repressed or -induced cells lacked the amorphous material and associated gold labeling. Bars represent 1 μm.

Article Snippet: Antibiotics were used at the following concentrations unless otherwise indicated: ampicillin, 50 μg/ml; chloramphenicol, 50 μg/ml; kanamycin, 50 μg/ml; nalidixic acid, 50 μg/ml; and rifampin, 50 μg/ml. table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 E. coli strain or plasmid Relevant characteristics Source or reference E. coli strains CFT073 Pyelonephritis isolate, fim + pap + hly + 31 CFT073 Rif Spontaneous rifampin-resistant mutant of CFT073 This study UMD100 CFT073 aufC ::pSSP104, ampicillin resistant This study UMD132 CFT073 Δ( aufABCDEFG ), nalidixic and chloramphenicol resistant This study UMD133 CFT073 Δ( aufABCDEFG ), nalidixic resistant This study ORN172 Δ( fimBEACDFGH ), kanamycin resistant 43 HB101 F − ( gpt-proA ) 62 leu supe44 ara-14 galK2 lacY1 ( mcrC-mrr ) rpsL20 xyl-5 mtl-1 recA13 37 TOP10F′ F′[ lacI q Tn 10 (Tet R )] mrcA Δ( mrr-hsdRMS-mrcrBC ) φ80 lacZ ΔM15 Δφ lacX74 recA1 araD139 Δ( ara-leu )7697 galU galk rpsL (Str r ) endA1 nupG Invitrogen BL21-AI F − ompT hsdS B (r B − m B − ) gal dcm (DE3) araB :: T7RNAP-tet A Invitrogen BL21 (DE3)pLysS F − ompT hsdS B (r B − m B − ) gal dcm (DE3) pLysS (Cam r ) Invitrogen S17-λ pir pro thi recA hsdR; chromosomal RP4-2 (Tn 1 ::ISR 1 tet ::Mu Km::Tn 7 ); λpir 17 Plasmids pcosmid 12A Cosmid clone of CFT073 that contains the entire auf operon pELB1 pCRScript Cam (SK+) with the 8.6-kb BsiWI/KasI fragment containing the auf operon This study pELB2 pBlueScript-II KS(+) with the 9-kb NotI/XhoI fragment containing auf operon under T7 promoter control This study pAufA pCR T7/NT-TOPO with the aufA gene lacking the codons for the signal sequence This study pPCRScript Cam (SK+) Cloning vector, chloramphenicol resistant Stratagene pBlueScript-II KS (+) Cloning vector, ampicillin resistant Stratagene pCR T7/NT-TOPO Cloning vector, ampicillin resistant Invitrogen pGP704 Suicide vector, ampicillin resistant, R6K origin 29 pT-Aadv Cloning vector, ampicillin resistant Clonetech pSSP102 pT-Adv with the 1.2-kb aufC fragment Stratagene pSSP104 pGP704 with the 1.2-kb XabI/KpnI aufC fragment, ampicillin resistant This study PKD46 Low-copy-number plasmid encoding the phage λ Red recombinase expressing γ, β, and exo from the arabinose-inducible Para B 8 pFT-A Helper plasmid, carrying the FLP recombinase, ampicillin resistant 34 pKD3 Plasmid containing an FRT-flanked cat gene, ampicillin and chloramphenicol resistant 8 Open in a separate window Strains and plasmids used in this study Recombinant DNA methods.

Techniques: Immuno-Electron Microscopy, Expressing, Plasmid Preparation, Negative Staining, Labeling