cloning vector pbluescript ks (ap r  (Agilent technologies)

Journal:

Article Title: A Vibrio vulnificus Type IV Pilin Contributes to Biofilm Formation, Adherence to Epithelial Cells, and Virulence

doi: 10.1128/IAI.73.3.1411-1422.2005

Figure Lengend Snippet: Bacterial strains and plasmids used in this study

Article Snippet: Isopropyl -β- d -thiogalactopyranoside (IPTG) was used at a final concentration of 0.2 or 1 mM. table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Strains, vector, or plasmid Relevant characteristic(s) Source or reference Strains V. vulnificus C7184/OP Wild type (clinical isolate) 73 C7184DΩ C7184 with disrupted pilD This laboratory C7184AΩ C7184 with disrupted pilA This study MO6-24/OP Wild type (clinical isolate) 34 PAC1 Wild type (environmental isolate) M. Coyle P. aeruginosa PAK-NP Nonpiliated mutant 46 PAK-NP(pRP383) Nonpiliated mutant expressing PilA from V. vulnificus This study E. coli DH5α supE44lacU169 (Δ lacZ ΔM15) hsdR17 recA lendA1gyrA96 thi-1 relA1 BRL SM10-λpir thi thr leu tonAlacY supE recA [RP4-2-Tc::Mu] λpir R6K Km r 51 BL21 F − ompT hsd S B (r B − m B − ) gal dcm 61 , 62 pFLAG-MAC Ap r FLAG fusion cloning vector Sigma pFLAG-ATS Ap r FLAG fusion cloning vector Sigma Cloning vectors pZErO-2 Kan r Zero background cloning vector Invitrogen pMMB67EH.cam Cm r Ap r broad-host-range cloning vector, lacI q / tac promoter 17 pBluescript II KS + Ap r phagemid cloning vector Stratagene pEP185.2 Cm r suicide vector 22 Recombinant plasmids pRP-P38 9- to 10-kb PstI fragment from C7184 carrying the pilABCD cluster cloned into pZErO-2 This study pRP102 2.04-kb PCR-generated fragment encompassing pilA cloned into pKS + This study pRP102Ω 2.04-kb PCR-generated fragment encompassing pilA, disrupted with the 2.0-kb Ω interposon, cloned into pEP185.2 This study pRP383 624-bp XhoI-HindIII fragment encompassing pilA cloned into pMMB67EH.cam This study pRPA110 PCR-generated pilA cloned into pFLAG-MAC This study pRPA111 PCR-generated pilA cloned into pFLAG-ATS This study Open in a separate window Bacterial strains and plasmids used in this study DNA manipulations.

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

Journal:

Article Title: Genetic and Functional Analysis of the tbc Operons for Catabolism of Alkyl- and Chloroaromatic Compounds in Burkholderia sp. Strain JS150

doi: 10.1128/AEM.67.10.4805-4816.2001

Figure Lengend Snippet: Bacterial strains and plasmids used in this study

Article Snippet: Cultures maintained in liquid or solid media were incubated at 37°C. table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Strain or plasmid Relevant characteristic(s) a Source or reference Strains E. coli JM109 recA1 relA1 thi-1 Δ( lac-proAB ) gyrA96 hsdR17 endA1 supE44 (r K − m K + ) [F′ traD36 proAB lacI q Z Δ M15 ] Promega E. coli BL21 F − dcm ompT hsdR (r B − m B + ) gal Stratagene Burkholderia sp. strain JS150 Tol + Ben + Chlben + Crl + Phl + 15 P. aeruginosa PAO1c Prototroph 20 Plasmids pBluescript II KS + (SK + ) lacZ Ap r ; 2.96-kb cloning vector Stratagene pRO1727 Tc r Cb r ; 3.77-kb Pseudomonas cloning vector 8 pHYK2000 Cb r ; pRO1727::14.3-kb Hin dIII- Bam HI fragment from JS150 containing tbc1 and tbc2 This study pHYK2001 Cb r ; pRO1727::11.3-kb Hin dIII- Bam HI fragment of pHYK2000, with a 3-kb Hin dIII deletion This study pHYK2002 Cb r ; pRO1727::10.1-kb Hin dIII- Bam HI fragment of pHYK2000, with a 4.2-kb internal Not I deletion This study pHYK2003 Cb r ; pRO1727::8.9-kb Hin dIII- Bam HI fragment of pHYK2000, with a 5.4-kb internal Xho I deletion This study pHYK2004 Cb r ; pRO1727::10.5-kb Hin dIII- Xho I fragment of pHYK2000, with a 3.8-kb deletion fusing an internal Xho I site with a vector Sal I site This study pHYK2005 Cb r ; pRO1727::9.2-kb Bam HI- Xho I fragment of pHYK2000 ligated to Bam HI- and Xho I-digested pRO1727 This study pJCM1000 Ap r ; pBluescript::5.4-kb Xho I fragment of pHYK2000 This study pJCM1001 Ap r ; pBluescript::6.2-kb Hin dIII- Not I fragment of pHYK2000 This study Open in a separate window a Abbreviations: Tol, toluene; Ben, benzene; Chlben, chlorobenzene; Crl, cresol; Phl, phenol; Ap, ampicillin; Tc, tetracycline; Cb, carbenicillin.

Techniques: Plasmid Preparation, Clone Assay

Journal:

Article Title: Genetic and Functional Analysis of the tbc Operons for Catabolism of Alkyl- and Chloroaromatic Compounds in Burkholderia sp. Strain JS150

doi: 10.1128/AEM.67.10.4805-4816.2001

Figure Lengend Snippet: Physical and functional map of pHYK2000. (A) Restriction map of the 14.3-kb BamHI-HindIII DNA fragment from Burkholderia sp. strain JS150 cloned into vector pRO1727 as pHYK2000. The XhoI-HindIII fragment used as a probe is indicated. (B) Southern hybridization results for DNA digested with XhoI and HindIII. Lanes: C, control (cloned) DNA; Ch, chromosomal DNA; P, plasmid DNA. (C) Construction of deletion mutants of pHYK2000 and assays for substrates oxidized by each construct. For functional mapping of pHYK2000, HindIII, NotI, XhoI, BamHI-XhoI, and HindIII-XhoI deletions were constructed, and the constructs were used to assay for oxidation (+, weak; ++, intermediate; +++, strong) of toluene (Tol), benzene (Ben), and chlorobenzene (Chlb).

Article Snippet: Cultures maintained in liquid or solid media were incubated at 37°C. table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Strain or plasmid Relevant characteristic(s) a Source or reference Strains E. coli JM109 recA1 relA1 thi-1 Δ( lac-proAB ) gyrA96 hsdR17 endA1 supE44 (r K − m K + ) [F′ traD36 proAB lacI q Z Δ M15 ] Promega E. coli BL21 F − dcm ompT hsdR (r B − m B + ) gal Stratagene Burkholderia sp. strain JS150 Tol + Ben + Chlben + Crl + Phl + 15 P. aeruginosa PAO1c Prototroph 20 Plasmids pBluescript II KS + (SK + ) lacZ Ap r ; 2.96-kb cloning vector Stratagene pRO1727 Tc r Cb r ; 3.77-kb Pseudomonas cloning vector 8 pHYK2000 Cb r ; pRO1727::14.3-kb Hin dIII- Bam HI fragment from JS150 containing tbc1 and tbc2 This study pHYK2001 Cb r ; pRO1727::11.3-kb Hin dIII- Bam HI fragment of pHYK2000, with a 3-kb Hin dIII deletion This study pHYK2002 Cb r ; pRO1727::10.1-kb Hin dIII- Bam HI fragment of pHYK2000, with a 4.2-kb internal Not I deletion This study pHYK2003 Cb r ; pRO1727::8.9-kb Hin dIII- Bam HI fragment of pHYK2000, with a 5.4-kb internal Xho I deletion This study pHYK2004 Cb r ; pRO1727::10.5-kb Hin dIII- Xho I fragment of pHYK2000, with a 3.8-kb deletion fusing an internal Xho I site with a vector Sal I site This study pHYK2005 Cb r ; pRO1727::9.2-kb Bam HI- Xho I fragment of pHYK2000 ligated to Bam HI- and Xho I-digested pRO1727 This study pJCM1000 Ap r ; pBluescript::5.4-kb Xho I fragment of pHYK2000 This study pJCM1001 Ap r ; pBluescript::6.2-kb Hin dIII- Not I fragment of pHYK2000 This study Open in a separate window a Abbreviations: Tol, toluene; Ben, benzene; Chlben, chlorobenzene; Crl, cresol; Phl, phenol; Ap, ampicillin; Tc, tetracycline; Cb, carbenicillin.

Techniques: Functional Assay, Clone Assay, Plasmid Preparation, Hybridization, Construct

Journal:

Article Title: Effects of Combination of Different -10 Hexamers and Downstream Sequences on Stationary-Phase-Specific Sigma Factor ? S -Dependent Transcription in Pseudomonas putida

doi:

Figure Lengend Snippet: Bacterial strains and plasmids used in this study

Article Snippet: Antibiotics were added at the following final concentrations: ampicillin at 100 μg/ml for E. coli ; carbenicillin at 1,500 μg/ml, kanamycin at 50 μg/ml, and tetracycline at 10 μg/ml for P. putida . table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Strain or plasmid Genotype or construction Source or reference Strains E. coli TG1 supE hsdΔ5 thi Δ( lac-proAB ) F′ ( traD36 proAB + lacI q lacZ ΔM15) 6 BL21(DE3) hsdS gal (λcI ts 857 ind 1 Sam 7 nin 5 lac UV5-T7 gene 1 39 S17-1 λpir Tp r Sm r recA thi pro (r − m + ) RP4::2 Tc::Mu::Km Tn 7 λpir 30 C118 λpir Δ( ara-leu ) araD ΔlacX74 galE galK phoA20 thi-1 rpsE rpoB argE (Am) recA1 λpir phage lysogen 16 P. putida PaW85 Tn 4652 3 PKS54 Tn 4652 rpoS ::Km r This work PKSRpoS P. putida PKS54 rpoS under control of Ptac promoter and lacI q repressor; Tc r Plasmids a pBluescript KS(+) Cloning vector (Ap r ) Stratagene pBlcrpoS-I pBluescript KS(+) containing 1-kb PCR-amplified rpoS cloned into Eco RV site pUC4K Cloning vector containing Km r gene from transposon Tn 903 (Ap r Km r ) 34 , 46 pBlcrpoS-Km r 2.5-kb rpoS -Km r sequence containing Xba I- Eco RI fragment from pUC4K cloned into Eco 72I site in rpoS gene This work pUTmini.Tn 5 luxAB Delivery plasmid for mini-Tn 5 luxAB (Ap r Tet r ) 8 pUTrpoS-Km r 2.5-kb rpoS -Km r sequence-containing Xba I- Eco RI fragment from pBlcrpoS-Km r cloned into pUTmini-Tn 5 luxAB This work pBlcrpoS-II pBluescript KS(+) containing 900-bp PCR-amplified rpoS cloned into Eco RV site This work pET24d Protein expression vector (Km r ) Stratagene pET24d-rpoS pET24d containing rpoS gene from pBlcrpoS-II inserted into Nco I and Hin dIII sites This work pKTlacZ Cloning vector (Ap r ) 18 pBRlacItac Ptac promoter and lacI q repressor in 2.2-kb Nru I- Eco RI fragment from plasmid pMMB208 cloned into Eco RV- Eco RI-cleaved pBR322 — b pMir61 rpoS gene of P. putida KT2440 in pUN19Ø 36 pBRlacItac-rpoS rpoS in 1-kb Xho I- Hin dIII fragment from plasmid pMir61 cloned into Sal I- Sma I-cleaved pBRlacItac This work pUC18Not pUC18 with Not I restriction site in multicloning region (Km r ) 16 pUCptac-rpoS 3.2-kb rpoS expression cassette Ptac-rpoS-lacI q from pBRlacItac-rpoS inserted into Bam HI- Kpn I-cleaved pUC18Not This work pUTptac-rpoS Ptac-rpoS-lacI q from pUCptac-rpoS inserted into Not I-cleaved pUTmini-Tn 5 luxAB This work Open in a separate window a The strategy of cloning of the fusion promoters and their mutant forms into promoter probe vector pKTlacZ is shown in Materials and Methods and in Fig. and . b —, Hõrak and Kivisaar, submitted.

Techniques: Plasmid Preparation, Clone Assay, Sequencing, Expressing

Journal:

Article Title: Characterization and Role of tbuX in Utilization of Toluene by Ralstonia pickettii PKO1

doi:

Figure Lengend Snippet: Bacterial strains and plasmids used in this study

Article Snippet: Growth of liquid cultures and incubation of agar plates was carried out at 37°C except when cultures contained aromatic hydrocarbons, in which case incubations were done at 30°C. table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Strain or plasmid Relevant characteristics a Reference or source Strains E. coli DH5α F − φ80d lacZ ΔM15 Δ( lacZYA-argF ) U169 deoR recA1 endA1 phoA hsdR17 (r K − m K + ) supE44 λ − thi-1 gyrA96 relA1 15 JM109 recA1 relA1 thi Δ( lac-proAB ) gyrA96 hsdR17 endA1 supE44 Promega MM294 supE 44 hsdR endA1 pro thi D. Clewell R. pickettii PKO1 Tol + Phl + 41 P. aeruginosa PAO1c Prototroph 18 Plasmids pBluescript II KS + (SK + ) lacZ Ap r , 2.96-kb cloning vector Stratagene pKRZ1 Ap r Km r ; broad-host-range promoter probe vector containing lacZ as a reporter gene A. Chakrabarty pRO1727 Tc r Cb r ; 3.77-kb Pseudomonas cloning vector 8 pRO1614 Tc r Cb r ; 6-kb E. coli-Pseudomonas cloning vector 38 p352 X/S Ap r Km r ; pKRZ1:: 352-bp Xho I- Sma I fragment; PtbuA1-lacZ fusion 6 p3.4kb X/B Ap r Km r ; pKRZ1:: 3.4-kbp Xho I- Bgl II fragment; PtbuD-lacZ fusion 40 p454 X/S Ap r Km r ; pKRZ1:: 454-bp Xho I- Sma I fragment; PtbuX-lacZ fusion This study pRO1959 Tc s Cb r ; pRO1727:: Hin dIII- Bam HI (15.1-kbp) fragment of R. pickettii PKO1 containing tbuD , tbuA1UBVA2C , tbuT , and tbuX 24 pRO1966 Tc s Cb r ; pRO1727:: Cla I- Bam HI (10-kbp) fragment of pRO1959 containing tbuA1UBVA2C , tbuT , and tbuX 41 pHYK1000 Tc s Cb r ; pRO1727:: Cla I- Pvu II (1.96-kbp) fragment of pRO1966 containing tbuA1UBVA2C and tbuT This study pHYK1001 Tc r Cb s pRO1614:: Eco RI- Pvu II (3.1-kbp) fragment of pRO1966 containing tbuT 6 Open in a separate window a Abbreviations: Tol, toluene; Phl, phenol; Ap, ampicillin; Cb, carbenicillin; Tc, tetracycline; Km, kanamycin.

Techniques: Plasmid Preparation, Clone Assay

Journal:

Article Title: Characterization and Role of tbuX in Utilization of Toluene by Ralstonia pickettii PKO1

doi:

Figure Lengend Snippet: Expression of putative tbuX promoter region a

Article Snippet: Growth of liquid cultures and incubation of agar plates was carried out at 37°C except when cultures contained aromatic hydrocarbons, in which case incubations were done at 30°C. table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Strain or plasmid Relevant characteristics a Reference or source Strains E. coli DH5α F − φ80d lacZ ΔM15 Δ( lacZYA-argF ) U169 deoR recA1 endA1 phoA hsdR17 (r K − m K + ) supE44 λ − thi-1 gyrA96 relA1 15 JM109 recA1 relA1 thi Δ( lac-proAB ) gyrA96 hsdR17 endA1 supE44 Promega MM294 supE 44 hsdR endA1 pro thi D. Clewell R. pickettii PKO1 Tol + Phl + 41 P. aeruginosa PAO1c Prototroph 18 Plasmids pBluescript II KS + (SK + ) lacZ Ap r , 2.96-kb cloning vector Stratagene pKRZ1 Ap r Km r ; broad-host-range promoter probe vector containing lacZ as a reporter gene A. Chakrabarty pRO1727 Tc r Cb r ; 3.77-kb Pseudomonas cloning vector 8 pRO1614 Tc r Cb r ; 6-kb E. coli-Pseudomonas cloning vector 38 p352 X/S Ap r Km r ; pKRZ1:: 352-bp Xho I- Sma I fragment; PtbuA1-lacZ fusion 6 p3.4kb X/B Ap r Km r ; pKRZ1:: 3.4-kbp Xho I- Bgl II fragment; PtbuD-lacZ fusion 40 p454 X/S Ap r Km r ; pKRZ1:: 454-bp Xho I- Sma I fragment; PtbuX-lacZ fusion This study pRO1959 Tc s Cb r ; pRO1727:: Hin dIII- Bam HI (15.1-kbp) fragment of R. pickettii PKO1 containing tbuD , tbuA1UBVA2C , tbuT , and tbuX 24 pRO1966 Tc s Cb r ; pRO1727:: Cla I- Bam HI (10-kbp) fragment of pRO1959 containing tbuA1UBVA2C , tbuT , and tbuX 41 pHYK1000 Tc s Cb r ; pRO1727:: Cla I- Pvu II (1.96-kbp) fragment of pRO1966 containing tbuA1UBVA2C and tbuT This study pHYK1001 Tc r Cb s pRO1614:: Eco RI- Pvu II (3.1-kbp) fragment of pRO1966 containing tbuT 6 Open in a separate window a Abbreviations: Tol, toluene; Phl, phenol; Ap, ampicillin; Cb, carbenicillin; Tc, tetracycline; Km, kanamycin.

Techniques: Expressing

Journal:

Article Title: Regulation of the Transposase of Tn 4652 by the Transposon-Encoded Protein TnpC

doi:

Figure Lengend Snippet: Bacterial strains and plasmids

Article Snippet: P. putida was electrotransformed according to the protocol described by Sharma and Schimke ( 29 ). table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Strain or plasmid Genotype or construction Source or reference E. coli TG1 supE hsd Δ5 thi Δ( lac-proAB ) F′ ( traD36 proAB + lacI q lacZ ΔM15) 6 BL21(DE3) hsdS gal (λ c I ts 857 ind 1 S am7 nin 5 lac UV5-T7 gene 1 ) 32 P. putida PaW85 Tn 4652 4 PRS2000 Tn 4652 free 34 Plasmids a pBluescript KS Cloning vector (Ap r ) Stratagene pET19b Protein expression vector (Ap r ) Stratagene pET19-tnpA tnpA is fused with histidine tag in pET19b This work pKT240 Cloning vector (Ap r Km r ) 3 pEST1354 Plasmid containing Tn 4652 upstream of the pheBA operon 17 pKTtnpA(D/H) Tn 4652 tnpA gene within the 3.2-kb Dra I- Hin dIII fragment cloned into pKT240 This work (Fig. ) pKTtnpA(D/P) Tn 4652 tnpA and tnpC genes within the 3.7-kb Dra I- Pvu I fragment cloned into pKT240 This work (Fig. ) pKTtnpA(D/P) * pKTtnpA(D/P) with the tnpC gene disrupted by frameshift This work pKTGC/tnpA pKT240 plus pDEL2-GC promoter region plus the promoterless tnpA gene This work pKTGC/tnpAC pKT240 plus pDEL2-GC promoter region plus the promoterless tnpA gene with tnpC This work pGUS102 Promoter probe vector containing the gusA gene cloned as a 1.8-kb Eco RI fragment into pBR322 A. Eriksson pKTGUS Vector for translational fusions containing the gusA gene without translation initiation codon ATG in pKT240; Hin dIII restriction site designed at the 5′ end of the gusA is suitable for in-frame cloning This work pTr1 PDEL2-GC promoter region plus 42 nt of the coding region of tnpA fused with gusA in pKTGUS This work (Fig. B) pTr2 PDEL2-GC promoter region plus 546 nt of the coding region of tnpA fused with gusA in pKTGUS This work (Fig. B) pTr3 PDEL2-GC promoter region plus 1,166 nt of the coding region of tnpA fused with gusA in pKTGUS This work (Fig. B) pKT-ACG gusA is cloned downstream of tnpC in pKTtnpA(D/P) This work (Fig. B) pKT-AdelCG Cla I- Cfr 10I deletion derivative of pKT-ACG This work (Fig. B) pKT-a1CG Dra I- Cla I deletion derivative of pKT-ACG This work (Fig. B) pKT-a2CG Dra I- Nru I deletion derivative of pKT-ACG This work (Fig. B) pKT-CG Dra I- Cfr 10I deletion derivative of pKT-ACG This work (Fig. B) Open in a separate window a Oligonucleotides used for construction of the plasmids are described in Materials and Methods.

Techniques: Plasmid Preparation, Clone Assay, Expressing